Gene concerning brassinosteroid-sensitivity of plants and utilization thereof

ABSTRACT

The present inventors successfully produced rice dwarf mutant d61 and also isolated the OsBRI1 gene which corresponds to a region in the d61 locus. OsBRI1 is found to increase plant brassinosteroid sensitivity. Moreover, the present inventors showed that OsBRI1 functions in growth and development process of rice, such as, internode elongation by inducing internode cell elongation and the inclination of the lamina joint. By introducing antisense nucleotides or dominant negative of OsBRI1, the present inventors produced transgenic rice plants whose phenotype was modified.

TECHNICAL FIELD

[0001] The present invention relates to a novel gene involved in plantbrassinosteroid sensitivity, the protein encoded by the gene, andproduction and use of the same.

BACKGROUND ART

[0002] Research in plant molecular biology has advanced dramatically inrecent years and is necessary for the analysis of various physiologicalphenomena. Dwarfism caused by artificial modification of grass type,especially the control of elongation growth, prevents plants fromlodging due to overgrowth caused by over fertilization. This preventionof lodging was demonstrated in Mexican wheat during the “GreenRevolution” and in miracle rice (IR-8) developed by the InternationalRice Research Center. Furthermore, in the case of cultivation at highdensity, such as rice cultivation, yields are expected to increase as aresult of the increase in the amount of sun light each plant receivesdue to the formation of upright leaves. Moreover, these modificationsare very important breeding targets because they may result in yieldincreases and also increase the efficiency of plant growth maintenance.However, current breeding methods cannot artificially modify plantmorphology.

[0003] Dwarfism is an abnormal growth caused by mutation in genesinvolved in controlling normal elongation growth. Plant elongationgrowth is the result of accumulation of cell division and cellelongation. Cell division and cell elongation are controlled by complexeffects caused by various factors, such as, exogenous environmentalfactors including temperature and light and endogenous environmentalfactors including plant hormones. Therefore, it is predicted that manygenes, such as those related to plant hormone biosynthesis and hormonereceptors directly and those related to the control of the expression ofthese genes, are involved in the dwarfism (Sakamoto et al. (2000) Kagakuto Seibutsu, 38: 131-139).

[0004] Almost all modern cultivars of japonica rice develop 15-16phytomers, consisting of leaves, axillary buds, and short or elongatedinternodes, during the vegetative stage. After the shoot meristem shiftsfrom the vegetative to the reproductive phase, the reproductive meristemdevelops about 10 phytomers consisting of undeveloped leaf, an elongatedinternode, and an axillary which develops into the primary rachisbranch. The phytomers formed in the vegetative stage can be classifiedinto three types in terms of the morphology of the internode (Suetsugu,Isao. (1968) Japan. J. Crop Sci. 37, 489-498). The first type isdeveloped in the juvenile phase and form undifferentiated nodes andinternodes. After the shoot apical meristem (SAM) shifts from thejuvenile to the adult phase, the nodal plate of the second typedifferentiates and the central part of the internode thereof decays toproduce an air space. The third type contains long elongated internodesas a result of growth from the intercalary meristem.

[0005] Phytomers of type 1 are produced first during vegetativedevelopment, followed by type 2 and then type 3 phytomers. Under normalgrowth conditions, the number of phytomers of each type in many japonicacultivars is 4-5, 6-7, and 4-5. The transition from type 1 to type 2 isstrictly regulated. After the serial development of 4-5 type 1phytomers, depending on the cultivar, the SAM develops type 2 phytomers.However, the transition from type 2 to type 3 does not depend on thenumber of development of type 2 phytomers. The SAM develops 15-16phytomers and shifts from the vegetative to the reproductive stage, thetype 3 phytomers then start to develop, and the uppermost four or fiveinternodes thereof start to elongate. If the timing of the transition ischanged by unusual growth conditions, the number of the type 2 phytomersalways affected thereby, but the number of the type 3 phytomer withelongated internode is unchanged (Suetsugu, isao. (1968) Japan. J. CropSci: 37, 489-498). This indicates that the transition of the SAM fromthe vegetative to the reproductive phase in rice induces internodeelongation, as well as in Arabidopsis.

[0006] However, there is an important difference between rice andArabidopsis. The elongated internodes in rice are derived from thevegetative SAM while those in Arabidopsis come from the reproductiveSAM. In rice, the uppermost four or five internodes develop from thevegetative SAM and initially are indistinguishable from the lower type 2internodes. When the SAM shifts to the reproductive phase,differentiation into type 3 internodes occurs due to the development ofintercalary meristems in the internodes. This synchronicity between thephase change of the SAM and the development of the intercalary meristemleads to the possibility that these processes might be linked by asignal coming from the SAM to the uppermost four or five phytomers whenits phase change occurs.

[0007] A large number of dwarf mutants of rice have been collected andcharacterized because of their agronomic importance. These dwarf mutantsare categorized into six groups based on the elongation pattern of theupper four to five internodes (FIG. 1; redrawn from Takeda, K. (1974)Bull. Fac. Agr. Hirosaki Univ. 22, 19-30. In rice, each internode isnumbered from top to bottom such that the uppermost internode just belowthe panicle is first). The present inventors can see that in the dn-typemutants the length of each internode is almost uniformly reduced,resulting in an elongation pattern similar to that of the wild typeplant. In contrast, the dm-type mutants show specific reduction of thesecond internode. Similar shortening of a specific internode is alsoobserved in the sh- and d6-type mutants, in which only the uppermostfirst internode or internodes below the uppermost are shortened,respectively. As these mutants with specifically shortened internodes,such as the dm-, d6-, and sh-types, might be defective in the perceptionof signals coming from the SAM, they should be especially useful for thestudy of the mechanism of internode elongation and its relationship tochanges in the SAM.

[0008] Brassinosteroids (BRs) are plant growth-promoting naturalproducts that are required for plant growth and development. There areonly a few reports on the physiological effects of brassinosteroids inthe growth and development of rice and other plants of the Gramineaefamily. Physiological researches indicate that exogenousbrassinosteroids alone, or in combination with auxin, enhance bending ofthe lamina joint in rice. The lamina joint has been used for a sensitivebioassay of brassinosteroids (Maeda, E. (1965) Physiol. Plant. 18,813-827; Wada, K. et al. (1981) Plant and Cell Physiol. 22, 323-325;Takeno, K. and Pharis, R. P. (1982) Plant Cell Physiol. 23, 1275-1281),because of high sensitivity thereof to brassinosteroids. In etiolatedwheat seedlings treatment with brassinolide or its derivative,castasterone, stimulates unrolling of the leaf blades (Wada, K. et al.(1985) Agric. Biol. Chem. 49, 2249-2251). Treatment with low or highconcentrations of brassinosteroids promotes or inhibits the growth ofroots in rice, respectively (Radi, S. H. and Maeda, E. (1988) J. CropSci. 57, 191-198). Brassinosteroids also promote the germination of riceseeds (Yamaguchi, T. et al. (1987) Stimulation of germination in agedrice seeds by pre-treatment with brassinolide. In: Proceeding of thefourteenth annual plant growth regulator society of America MeetingHonolulu. (Cooke A R), pp. 26-27).

[0009] Although these results indicate only effects due to exogenousbrassinosteroids, not due to endogenous brassinosteroids, they dosuggest that endogenous brassinosteroids have an important role ingrowth and developmental processes in plants of the Gramineae family.

[0010] On the other hand, there is some apparent disagreement in theliterature as to whether brassinosteroids induce cell elongation inplants of the Gramineae family. That is, brassinolide treatment does notinduce elongation of the leaf sheath of rice (Yokota, T. and Takahashi,N. (1986) Chemistry, physiology and agricultural application ofbrassinolide and related steroids. In: Plant growth substances 1985.(Bopp M, Springer-Verlag, Berlin/Heidelberg/New York) pp.129-138), butit does induce elongation of the coleoptile and mesocotyl in maize (He,R. -Y. et al. (1991) Effects of brassinolide on growth and chillingresistance of maize seedlings. In: Brassinosteroids-Chemistry,Bioactivity and Applications ACS symposium series 474. (Cutler H G C,Yokota T, Adam G, American Chemical Society, Washington D.C.), pp.220-230).

[0011] As shown by brassinosteroids synthesis mutants orbrassinosteroids insensitive mutants that show severe dwarfism withabnormal development of organs, the function of brassinolide is known indicotyledonous plants.

[0012] However, little is known about the function of endogenousbrassinosteroids in monocotyledonous plants, such as rice or otherplants of the Gramineae family.

DISCLOSURE OF THE INVENTION

[0013] The object of the present invention is to provide novel genesinvolved in brassinosteroid sensitivity from plants, preferably frommonocotyledonous plants. Another object of the present invention is tomodify plant brassinosteroid sensitivity by controlling the expressionof the gene. The modification in plant brassinosteroid sensitivitycauses a change in plant morphology. The preferable embodiment of thepresent invention provides plants with erect leaves which become dwarfeddue to the suppression of internode elongation caused by decreasedbrassinosteroid sensitivity.

[0014] By treatment with mutagenesis agent, the present inventorsisolated a novel rice dwarf mutant strain d61 (d61-1 and d61-2) whichshowed lower brassinosteroid sensitivity and had shorter internodes thanwild type plants.

[0015] Linkage analysis indicated that the d61 locus was highly linkedto a gene region that was homologous to Arabidopsis BRI1. The presentinventors isolated the gene (OsBRI1), which was homologous toArabidopsis BRI1 gene, by screening of a rice genomic DNA library.Nucleotide sequence analysis of the OsBRI1 gene from d61-1 and d61-2mutants indicated that there were single nucleotide substitutionscausing amino acid substitutions at different sites in each d61 allele.

[0016] Moreover, in order to confirm that the OsBRI1 gene corresponds tothe d61 locus, the OsBRI1 gene was introduced into d61 mutants. As aresult, the OsBRI1 gene complimented the d61 phenotype and caused themutant strain to have a wild-type phenotype. Therefore, it was indicatedthat d61 mutants are caused by loss of function of the OsBRI1 gene.Phenotypic analysis of plants revealed that the OsBRI1 gene functions invarious growth and development processes of rice including internodeelongation caused by formation of intercalary meristem and induction ofinternode cell longitudinal elongation, inclination of the lamina joint,and skotomorphogenesis in the dark.

[0017] Moreover, in the case where transgenic rice plants with OsBRI1antisense nucleotide were produced, most transgenic plants producederect leaves during seedling growth. All of the transgenic plants showeddwarf phenotype of various levels. Plants transformed with OSBRI1 havingthe dominant negative phenotype showed the same result.

[0018] The present invention had been made in view of such findings, andrelates to a novel gene involved in plant brassinosteroid sensitivity,the protein encoded by the gene, and production and use of the same.Moreover, the present invention relates to the production of modifiedplant by controlling expression of the gene.

[0019] More specifically, this invention provides:

[0020] (1) a DNA encoding a protein comprising the amino acid sequenceof SEQ ID NO: 2;

[0021] (2) the DNA of (1), wherein the DNA is a cDNA or a genomic DNA;

[0022] (3) the DNA of (1), wherein the DNA comprises a coding region ofthe nucleotide sequence of SEQ ID NO: 1 or 3;

[0023] (4) a DNA encoding a protein which has 55% or more homology tothe amino acid sequence of SEQ ID NO: 2 and which is functionallyequivalent to a protein comprising the amino acid sequence of SEQ ID NO:2, the DNA being selected from the group consisting of

[0024] (a) a DNA encoding a protein comprising the amino acid sequenceof SEQ ID NO: 2 in which one or more amino acids are substituted,deleted, added, and/or inserted; and

[0025] (b) a DNA hybridizing under stringent conditions with a DNAcomprising the nucleotide sequence of SEQ ID NO: 1 or 3;

[0026] (5) the DNA of (4), wherein the DNA encodes a protein having afunction selected from the group consisting of a function of increasingbrassinosteroid sensitivity in a plant, a function of inducingelongation of internode cells of a stem of a plant, a function ofpositioning microtubules perpendicular to the direction of elongation inan internode of a stem of a plant, a function of suppressing elongationof an internode of a neck of a plant, and a function of increasinginclination of a lamina of a plant;

[0027] (6) the DNA of (4) or (5), wherein the DNA is derived from amonocotyledonous plant;

[0028] (7) the DNA of (6), wherein the DNA is derived from a plant ofthe Gramineae family;

[0029] (8) a DNA encoding an antisense RNA complementary to a transcriptof the DNA of any one of (1) to (7);

[0030] (9) a DNA encoding an RNA having ribozyme activity whichspecifically cleaves a transcript of the DNA of any one of (1) to (7);

[0031] (10) a DNA which encodes an RNA repressing expression of the DNAof any one of (1) to (7) due to co-suppression when expressed in a plantcell and which has 90% or more homology to the DNA of any one of (1) to(7);

[0032] (11) a DNA which encodes a protein having a dominant negativephenotype to that of a protein encoded by the DNA of any one of (1) to(7);

[0033] (12) a vector which comprises the DNA of any one of (1) to (7);

[0034] (13) a transformed cell which comprises the DNA of any one of (1)to (7) or the vector of (12);

[0035] (14) a protein encoded by the DNA of any one of (1) to (7);

[0036] (15) a method for producing the protein of (14) the methodcomprising the steps of culturing the transformed cell of (13) andrecovering an expressed protein from the transformed cell or a culturesupernatant thereof;

[0037] (16) a vector comprising the DNA of any one of (8) to (11);

[0038] (17) a transformed plant cell comprising the DNA of any one of(1) to (11) or the vector of (12) or (16);

[0039] (18) a transformed plant comprising the transformed plant cell of(17);

[0040] (19) a transformed plant which is a progeny or a clone of thetransformed plant of (18);

[0041] (20) a breeding material of the transformed plant of (18) or(19); and

[0042] (21) an antibody which binds to the protein of (14).

[0043] The present invention provides a DNA encoding the OsBRI1 proteinderived from rice. The nucleotide sequence of OsBRI1 cDNA is shown inSEQ ID NO: 1, the amino acid sequence of the protein encoded by the DNAis shown in SEQ ID NO: 2, and the nucleotide sequence of the genomic DNAof OsBRI1 is shown in SEQ ID NO: 3 (the genomic DNA of SEQ ID NO: 3consists of one exon with no intron).

[0044] The gene of the present invention causes a rice dwarf mutant(d61) which has short internodes and reduced brassinosteroid sensitivitycompared to the wild type. Therefore, it is possible to modify plantmorphology by controlling the expression of the OsBRI1 gene.

[0045] The preferable modification in plant morphology in the presentinvention includes dwarfism of plants by suppressing expression of theDNA of the present invention. Dwarfism of plants has great value inagriculture and horticulture. For example, reduction of height of plantscan reduce the tendency of plants to lodge and can thereby increase seedweights. Moreover, it is possible to increase the number of plantindividuals which can be planted per unit area by reducing height ofplants and by making plant shape per plant more compact. These plantmodifications have great value specifically in the production of cropssuch as rice, corn, wheat, and such. It is also possible to produceornamental plants with new aesthetic value by dwarfism of height or culmlength of plants. It is also possible to produce miniature vegetables orfruits with new commercial value, such as “bite-size”, by dwarfism ofthem. Other than for industrial plants, dwarfism is important forexperimental plants because, for example, dwarf plants are not only moreeasily handled but they also help utilize experimental space moreeffectively by decreasing cultivation space.

[0046] It is possible to consider that brassinosteroid sensitivity canbe increased in brassinosteroid low sensitive plants by expressing theDNA of the present invention in the plants. Thereby, the yield of wholeplants may be increased by growing taller plants. Thus, this will beespecially useful for increasing yield for whole feed crops.

[0047] DNA encoding the OsBRI1 protein of the present invention includesgenomic DNA, cDNA, and chemically synthesized DNA. A genomic DNA andcDNA can be prepared according to conventional methods known to thoseskilled in the art. More specifically, a genomic DNA can be prepared,for example, as follows: (1) extract genomic DNA from plant cells ortissues; (2) construct a genomic library (utilizing a vector, such asplasmid, phage, cosmid, BAC, PAC, and such); (3) spread the library; and(4) conduct colony hybridization or plaque hybridization using a probeprepared based on the DNA encoding a protein of the present invention(e.g., SEQ ID NO: 1 or 3). Alternatively, a genomic DNA can be preparedby PCR, using primers specific to a DNA encoding the protein of thepresent invention (e.g. SEQ ID NO: 1 or 3). On the other hand, cDNA canbe prepared, for example, as follows: (1) synthesize cDNAs based onmRNAs extracted from plant cells or tissues; (2) prepare a cDNA libraryby inserting the synthesized cDNA into vectors, such as XZAP; (3) spreadthe cDNA library; and (4) conduct colony hybridization or plaquehybridization as described above. Alternatively, cDNA can be alsoprepared by PCR.

[0048] The present invention includes DNAs encoding proteinsfunctionally equivalent to the OsBRI1 protein of SEQ ID NO: 2. Herein,the term “functionally equivalent to the OsBRI1 protein” means that theobject protein has equal functions to those of the OsBRI1 protein of SEQID NO: 2, such as, for example, a function of increasing brassinosteroidsensitivity in a plant, a function of inducing elongation of aninternode of a stem of a plant, a function of positioning microtubulesperpendicular to the direction of elongation in internode cells of astem of a plant, a function of suppressing elongation of an internode ofa neck of a plant, and/or a function of increasing inclination of alamina of a plant. Such DNA is derived preferably from monocotyledonousplants, more preferably from plants of the Gramineae family, and mostpreferably from rice.

[0049] Examples of such DNAs include those encoding mutants,derivatives, alleles, variants, and homologues comprising the amino acidsequence of SEQ ID NO: 2 wherein one or more amino acids aresubstituted, deleted, added, and/or inserted.

[0050] Examples of methods for preparing a DNA encoding a proteincomprising altered amino acids well known to those skilled in the artinclude the site-directed mutagenesis (Kramer, W. and Fritz, H. -J.(1987) “Oligonucleotide-directed construction of mutagenesis via gappedduplex DNA.” Methods in Enzymology, 154: 350-367). The amino acidsequence of a protein may also be mutated in nature due to the mutationof a nucleotide sequence. A DNA encoding proteins having the amino acidsequence of a natural OsBRI1 protein (SEQ ID NO: 2) wherein one or moreamino acids are substituted, deleted, and/or added are also included inthe DNA of the present invention, so long as they encode a proteinfunctionally equivalent to the natural OsBRI1 protein. Additionally,nucleotide sequence mutants that do not give rise to amino acid sequencechanges in the protein (degeneracy mutants) are also included in the DNAof the present invention. The number of nucleotide mutations of the DNAof interest corresponds to, at amino acid level, typically 100 residuesor less, preferably 50 residues or less, more preferably 20 residues orless, and still more preferably 10 residues or less (for example, 5residues or less, or 3 residues or less).

[0051] Whether a certain DNA actually encodes a protein which has afunction of increasing inclination of a lamina of a plant can beevaluated, for example, by performing a “lamina joint test” for plantsin which the expression of the DNA has been suppressed and by comparingthe results with those for wild-type plants (See Example 4). The resultof the test may also be an index for evaluating brassinosteroidsensitivity in a plant. In order to evaluate whether the DNA encodes aprotein which has a function of inducing elongation of an internode of astem of a plant, a function of positioning microtubules perpendicular tothe direction of elongation in internode cells of a stem of a plant, ora function of suppressing elongation of an internode of a neck of aplant, the morphology of the internode cell of the plant in whichexpression of the DNA has been suppressed can be observed to be comparedwith that of wild type (See Examples 2 and 3).

[0052] A DNA encoding a protein functionally equivalent to the OsBRI1protein described in SEQ ID NO: 2 can be produced, for example, bymethods well known to those skilled in the art including: methods usinghybridization techniques (Southern, E. M. (1975) Journal of MolecularBiology, 98, 503); and polymerase chain reaction (PCR) techniques(Saiki, R. K. et al. (1985) Science, 230, 1350-1354; Saiki, R. K. et al.(1988) Science, 239, 487-491). That is, it is routine for a personskilled in the art to isolate a DNA with high homology to the OsBRI1gene from rice and other plants using the OsBRI1 gene (SEQ ID NO: 1 or3) or parts thereof as a probe, and oligonucleotides hybridizingspecifically to the gene as a primer. Such DNA encoding proteinsfunctionally equivalent to the OsBRI1 protein, obtainable byhybridization techniques or PCR techniques, are included in the DNA ofthis invention.

[0053] Hybridization reactions to isolate such DNAs are preferablyconducted under stringent conditions. Stringent hybridization conditionsof the present invention include conditions such as: 6 M urea, 0.4% SDS,and 0.5×SSC; and those which yield a similar stringency with theconditions. DNAs with higher homology are expected to be isolatedefficiently when hybridization is performed under conditions with higherstringency, for example, 6 M urea, 0.4% SDS, and 0.1×SSC. Those DNAsisolated under such conditions are expected to encode a protein having ahigh amino acid level homology with OsBRI1 protein (SEQ ID NO: 2).Herein, “high homology” means an identity of at least 55% or more, morepreferably 70% or more, and most preferably 90% or more (e.g., 95% ormore), between full-length of amino acids.

[0054] The degree of homology of one amino acid sequence or nucleotidesequence to another can be determined by following the algorithm BLASTby Karlin and Altschul (Proc. Natl. Acad. Sci. USA, 90: 5873-5877,1993). Programs such as BLASTN and BLASTX were developed based on thisalgorithm (Altschul et al. J. Mol. Biol. 215: 403-410, 1990). To analyzea nucleotide sequences according to BLASTN based on BLAST, theparameters are set, for example, as score=100 and word length=12. On theother hand, parameters used for the analysis of amino acid sequences bythe BLASTX based on BLAST include, for example, score=50 and wordlength=3. Default parameters of each program are used when using BLASTand Gapped BLAST program. Specific techniques for such analysis areknown in the art (http://www.ncbi.nlm.nih.gov.) The DNA of the presentinvention can be used, for example, to prepare recombinant proteins,produce transformed plants with phenotypes altered by controllingexpression thereof as described above, and so on.

[0055] A recombinant protein is usually prepared by inserting a DNAencoding a protein of the present invention into an appropriateexpression vector, introducing said vector into an appropriate cell,culturing the transformed cells, and purifying expressed proteins. Arecombinant protein can be expressed as a fusion protein with otherproteins so as to be easily purified, for example, as a fusion proteinwith maltose binding protein in Escherichia coli (New England Biolabs,USA, vector pMAL series), as a fusion protein withglutathione-S-transferase (GST) (Amersham Pharmacia Biotech, vector pGEXseries), or tagged with histidine (Novagen, pET series). The host cellis not limited so long as the cell is suitable for expressing therecombinant protein. It is possible to utilize yeasts or various animal,plant, or insect cells besides the above described E. coli. A vector canbe introduced into a host cell by a variety of methods known to oneskilled in the art. For example, a transformation method using calciumions (Mandel, M. and Higa, A. (1970) Journal of Molecular Biology, 53,158-162; Hanahan, D. (1983) Journal of Molecular Biology, 166, 557-580)can be used to introduce a vector into E. coli. A recombinant proteinexpressed in host cells can be purified and recovered from the hostcells or the culture supernatant thereof by known methods. When arecombinant protein is expressed as a fusion protein with maltosebinding protein or other partners, the recombinant protein can be easilypurified by affinity chromatography.

[0056] The resulting protein can be used to prepare an antibody thatbinds to the protein. For example, a polyclonal antibody can be preparedby immunizing immune animals, such as rabbits, with a purified proteinof the present invention or its portion, collecting blood after acertain period, and removing clots. A monoclonal antibody can beprepared by fusing myeloma cells with the antibody-forming cells ofanimals immunized with the above protein or its portion, isolating amonoclonal cell expressing a desired antibody (hybridoma), andrecovering the antibody from the cell. The obtained antibody can beutilized to purify or detect a protein of the present invention.Accordingly, the present invention includes antibodies that bind toproteins of the invention.

[0057] In order to produce a transformed plant in which DNAs of thepresent invention are expressed, a DNA encoding a protein of the presentinvention is inserted into an appropriate vector; the vector is thenintroduced into a plant cell; and finally, the resulting transformedplant cell is regenerated.

[0058] On the other hand, a transformed plant with suppressed expressionof DNAs of the present invention can be created using DNA that repressesthe expression of a DNA encoding a protein of the present invention:wherein the DNA is inserted into an appropriate vector, the vector isintroduced into a plant cell, and then, the resulting transformed plantcell is regenerated. The phrase “suppression of expression of DNAencoding a protein of the present invention” includes suppression ofgene transcription as well as suppression of translation into protein.It also includes not only the complete inability of expression of DNAbut also reduction of expression.

[0059] The expression of a specific endogenous gene in plants can berepressed by methods utilizing antisense technology, the methods whichare commonly used in the art. Ecker et al. were the first to demonstratethe antisense effect of an antisense RNA introduced by electroporationin plant cells by using the transient gene expression method (J. R.Ecker and R. W. Davis (1986) Proc. Natl. Acad. Sci. USA 83: 5372).Thereafter, the target gene expression was reportedly reduced in tobaccoand petunias by expressing antisense RNAs (A. R. van der Krol et al.(1988) Nature 333: 866). The antisense technique has now beenestablished as a means to repress target gene expression in plants.

[0060] Multiple factors are required for antisense nucleic acid torepress the target gene expression. These include, inhibition oftranscription initiation by triple strand formation; suppression oftranscription by hybrid formation at the site where the RNA polymerasehas formed a local open loop structure; transcription inhibition byhybrid formation with the RNA being synthesized; suppression of splicingby hybrid formation at the junction between an intron and an exon;suppression of splicing by hybrid formation at the site of spliceosomeformation; suppression of mRNA translocation from the nucleus to thecytoplasm by hybrid formation with mRNA; suppression of splicing byhybrid formation at the capping site or at the poly A addition site;suppression of translation initiation by hybrid formation at the bindingsite for the translation initiation factors; suppression of translationby hybrid formation at the site for ribosome binding near the initiationcodon; inhibition of peptide chain elongation by hybrid formation in thetranslated region or at the polysome binding sites of mRNA; andsuppression of gene expression by hybrid formation at the sites ofinteraction between nucleic acids and proteins. These factors repressthe target gene expression by inhibiting the process of transcription,splicing, or translation (Hirashima and Inoue, “Shin Seikagaku JikkenKoza (New Biochemistry Experimentation Lectures) 2, Kakusan (NucleicAcids) IV, Idenshi No Fukusei To Hatsugen (Replication and Expression ofGenes)”, Nihon Seikagakukai Hen (The Japanese Biochemical Society),Tokyo Kagaku Dozin, pp. 319-347, (1993)).

[0061] An antisense sequence of the present invention can repress thetarget gene expression by any of the above mechanisms. In oneembodiment, if an antisense sequence is designed to be complementary tothe untranslated region near the 5′ end of the gene's mRNA, it willeffectively inhibit translation of a gene. It is also possible to usesequences complementary to the coding regions or to the untranslatedregion on the 3′ side. Thus, the antisense DNA used in the presentinvention includes DNA having antisense sequences against both theuntranslated regions and the translated regions of the gene. Theantisense DNA to be used is connected downstream from an appropriatepromoter, and, preferably, a sequence containing the transcriptiontermination signal is connected on the 3′ side. The DNA thus preparedcan be transfected into the desired plant by known methods. The sequenceof the antisense DNA is preferably a sequence complementary to theendogenous gene of the plant to be transformed or a part thereof, but itneed not be perfectly complementary so long as it can effectivelyinhibit the gene expression. The transcribed RNA is preferably at least90%, and most preferably at least 95% complementary to the transcribedproducts of the target gene. Sequence complementarity may be determinedusing the above-described search.

[0062] In order to effectively inhibit the expression of the target geneby means of an antisense sequence, the antisense DNA should be at least15 nucleotides long, preferably at least 100 nucleotides long, and morepreferably at least 500 nucleotides long. The antisense DNA to be usedis generally shorter than 5 kb, and preferably shorter than 2.5 kb.

[0063] DNA encoding ribozymes can also be used to repress the expressionof endogenous genes. A ribozyme is an RNA molecule that has catalyticactivity. There are many ribozymes having various activities. Researchon ribozymes as RNA cleaving enzymes has enabled the design of aribozyme that site-specifically cleaves RNA. While some ribozymes of thegroup I intron type or the mRNA contained in RNaseP consist of 400nucleotides or more, others belonging to the hammerhead type or thehairpin type have an activity domain of about 40 nucleotides (MakotoKoizumi and Eiko Ohtsuka, (1990) Tanpakushitsu Kakusan Kohso (Nucleicacid, Protein, and Enzyme), 35: 2191).

[0064] The self-cleavage domain of a hammerhead type ribozyme cleaves atthe 3′ side of C15 of the sequence G13U14C15. Formation of a nucleotidepair between U14 and A at the ninth position is considered important forthe ribozyme activity. Furthermore, it has been shown that the cleavagealso occurs when the nucleotide at the 15th position is A or U insteadof C (M.. Koizumi et al., (1988) FEBS Lett. 228: 225). If the substratebinding site of the ribozyme is designed to be complementary to the RNAsequences adjacent to the target site, one can create arestriction-enzyme-like RNA cleaving ribozyme which recognizes thesequence UC, UU, or UA within the target RNA (M. Koizumi et al., (1988)FEBS Lett. 239: 285; Makoto Koizumi and Eiko Ohtsuka, (1990)Tanpakushitsu Kakusan Kohso (Protein, Nucleic acid, and Enzyme), 35:2191; M. Koizumi et al., (1989) Nucleic Acids Res. 17: 7059). Forexample, in the coding region of the OsBRI1 gene (SEQ ID NO: 1 or 3),there is a plurality of sites that can be used as the ribozyme target.

[0065] The hairpin type ribozyme is also useful in the presentinvention. A hairpin type ribozyme can be found, for example, in theminus strand of the satellite RNA of tobacco ringspot virus (J. M.Buzayan, Nature 323: 349 (1986)). This ribozyme has also been shown totarget-specifically cleave RNA (Y. Kikuchi and N. Sasaki, (1992) NucleicAcids Res. 19: 6751; Yo Kikuchi, (1992) Kagaku To Seibutsu (Chemistryand Biology) 30: 112).

[0066] The ribozyme designed to cleave the target is fused with apromoter, such as the cauliflower mosaic virus 35S promoter, and with atranscription termination sequence, so that it will be transcribed inplant cells. However, if extra sequences have been added to the 5′ endor the 3′ end of the transcribed RNA, the ribozyme activity can be lost.In this case, one can place an additional trimming ribozyme, whichfunctions in cis to perform the trimming on the 5′ or the 3′ side of theribozyme portion, in order to precisely cut the ribozyme portion fromthe transcribed RNA containing the ribozyme (K. Taira et al. (1990)Protein Eng. 3: 733; A. M. Dzaianott and J. J. Bujarski (1989) Proc.Natl. Acad. Sci. USA 86: 4823; C. A. Grosshands and R. T. Cech (1991)Nucleic Acids Res. 19: 3875; K. Taira et al. (1991) Nucleic Acid Res.19: 5125). Multiple sites within the target gene can be cleaved byarranging these structural units in tandem to achieve greater effects(N. Yuyama et al. (1992) Biochem. Biophys. Res. Commun. 186: 1271). Byusing such ribozymes, it is possible to specifically cleave thetranscripts of the target gene in the present invention, therebyrepressing the expression of said gene.

[0067] Endogenous gene expression can also be repressed byco-suppression through the transformation by DNA having a sequenceidentical or similar to the target gene sequence. “Co-suppression”refers to the phenomenon in which, when a gene having a sequenceidentical or similar to the target endogenous gene sequence isintroduced into plants by transformation, expression of both theintroduced exogenous gene and the target endogenous gene becomesrepressed. Although the detailed mechanism of co-suppression is unknown,it is frequently observed in plants (Curr. Biol. (1996) 7: R793 (1997),Curr. Biol. 6:810). For example, if one wishes to obtain a plant body inwhich the OsBRI1 gene is co-repressed, the plant in question can betransformed with a vector DNA designed so as to express the OsBRI1 geneor DNA having a similar sequence to select a plant having the OsBRI1mutant character, e.g., a plant with suppressed internode elongation,among the resultant plants. The gene to be used for co-suppression doesnot need to be completely identical to the target gene, but it shouldhave at least 70% or more sequence identity, preferably 80% or moresequence identity, and more preferably 90% or more (e.g., 95% or more)sequence identity. Sequence identity may be determined byabove-described search.

[0068] In addition, endogenous gene expression in the present inventioncan also be repressed by transforming the plant with a gene having thedominant negative phenotype of the target gene. Herein, “a DNA encodingthe protein having the dominant negative phenotype” refers to a DNAencoding a protein which, when the DNA is expressed, can eliminate orreduce the activity of the protein encoded by the endogenous gene of thepresent invention inherent to the plant. Preferably, it is a DNAencoding the peptide (e.g., peptide which contains from 739 to 1035residues of amino acids of SEQ ID NO: 2 or peptides of another proteinequivalent to the peptide) which lacks the N-terminal region butcontains the kinase region of the protein of the present invention.Whether the DNA of interest has the function to eliminate or enhanceactivity of the endogenous gene of the present invention can bedetermined, as mentioned above, by whether the DNA of interesteliminates or reduces a function of increasing brassinosteroidsensitivity in a plant, a function of inducing elongation of aninternode of a stem of a plant, a function of positioning microtubulesperpendicular to the direction of elongation in internode cells of astem of a plant, a function of suppressing elongation of an internode ofa neck of a plant, and/or a function of increasing inclination of alamina of a plant.

[0069] Vectors used for the transformation of plant cells are notlimited as long as the vector can express inserted genes in plant cells.For example, vectors comprising promoters for constitutive geneexpression in plant cells (e.g., califlower mosaic virus 35S promoter);and promoters inducible by exogenous stimuli can be used. The term“plant cell” used herein includes various forms of plant cells, such ascultured cell suspensions, protoplasts, leaf sections, and callus.

[0070] A vector can be introduced into plant cells by known methods,such as the polyethylene glycol method, electroporation, Agrobacteriummediated transfer, and particle bombardment. Plants can be regeneratedfrom transformed plant cells by known methods depending on the type ofthe plant cell (Toki et al., (1995) Plant Physiol. 100:1503-1507). Forexample, transformation and regeneration methods for rice plantsinclude: (1) introducing genes into protoplasts using polyethyleneglycol, and regenerating the plant body (suitable for indica ricecultivars) (Datta, S. K. (1995) in “Gene Transfer To Plants”, Potrykus Iand Spangenberg Eds., pp66-74); (2) introducing genes into protoplastsusing electric pulse, and regenerating the plant body (suitable forjaponica rice cultivars) (Toki et al (1992) Plant Physiol. 100,1503-1507); (3) introducing genes directly into cells by the particlebombardment, and regenerating the plant body (Christou et al. (1991)Bio/Technology, 9: 957-962); (4) introducing genes using Agrobacterium,and regenerating the plant body; and so on. These methods are alreadyestablished in the art and are widely used in the technical field of thepresent invention. Such methods can be suitably used for the presentinvention.

[0071] Once a transformed plant, wherein the DNA of the presentinvention is introduced into the genome, is obtained, it is possible togain descendants from that plant body by sexual or vegetativepropagation. Alternatively, plants can be mass-produced from breedingmaterials (for example, seeds, fruits, ears, tubers, tubercles, tubs,callus, protoplast, etc.) obtained from the plant, as well asdescendants or clones thereof. Plant cells transformed with the DNA ofthe present invention, plant bodies including these cells, descendantsand clones of the plant, as well as breeding materials obtained from theplant, its descendant and clones, are all included in the presentinvention.

[0072] The plant of the present invention is preferably amonocotyledonous plant, more preferably a plant of the Gramineae family,and most preferably a rice. The phenotype of the plant of the presentinvention is different from the wild type phenotype. The phenotypeschanged in the plants developed by the present invention includebrassinosteroid sensitivity of a plant, plant growth such as internodecell elongation of the plant stem and internode elongation of the ear,inclination of leaves, and the positioning of microtubules perpendicularto the direction of internode cell elongation in the plant stem.

BRIEF DESCRIPTION OF THE DRAWINGS

[0073]FIG. 1 shows a schematic diagram of the internode elongationpattern of wild type rice and various dwarf mutant and wild-type riceplants. The relative lengths of the each internode to the stem are shownin the schematic diagram. Wild type is shown as N.

[0074]FIG. 2 represents photographs which show the phenotype of the d61mutants.

[0075] (A) Gross morphology. (Left) Wild type plant; (centre) d61-1mutant (weak allele); (right) d61-2 mutant (strong allele).

[0076] (B) Elongation pattern of internodes. The wild type plant (left)shows the N-type of the elongation pattern, while the d61-1 (centre) andd61-2 (right) mutants show typical dn- and d6-type patterns,respectively.

[0077] (C) Panicle structure. The wild type plant (left) has a shortpanicle, while the d61-1 (centre) and d61-2 (right) mutants have longerpanicles.

[0078] (D) Erect leaf of d61. The leaves of the wild type plant (left)are bent at the lamina joint indicated by the white arrow, while theleaves of d61-1 (centre) and d61-2 (right) mutants are more erect.

[0079] (E) Leaf sheath of d61. The leaf sheath in the d61-1 (centre) andd61-2 (right) mutants is shorter than in the wild type plants (left).

[0080]FIG. 3 represents microphotographs which show the structure ofwell-developed internodes from wild type and d61-2 rice plants, asfollows:

[0081] (A) longitudinal sections of the first internodes from wild type;

[0082] (B) longitudinal sections of the second internodes from wildtype;

[0083] (C) longitudinal sections of the third internodes from wild type;

[0084] (D) longitudinal sections of the fourth internodes from wildtype;

[0085] (E) longitudinal sections of the first internodes from d61-2 riceplants;

[0086] (F) longitudinal sections of the second internodes from d61-2rice plants;

[0087] (G) longitudinal sections of the third internodes from d61-2 riceplants; and

[0088] (H) longitudinal sections of the fourth internodes from d61-2rice plants.

[0089] Bar=100 μm, respectively.

[0090]FIG. 4 are photographs and drawings which show the orientation ofmicrotubules in elongating sells in the first internode of wild type andd61-2 plants, consisting of immunofluorescence images (A and B) orschematic presentation (C and D) of the microtubule arrangement ininternodal parenchyma cells of the first internode from wild type (A andC) and d61-2 (B and D) plants. Bars=50 μm.

[0091]FIG. 5 is a photograph which shows the response of seedlings ofwild plant, D61-1, and d61-2 to brassinolide.

[0092] Seeds were germinated on agar plates in the presence or absenceof 1 μM brassinolide (BL). Seedlings were observed 1 day aftergermination. BL treatment induced abnormal growth in wild plant, whilemutant seedlings were not affected thereto.

[0093]FIG. 6 is a photograph which shows effect of brassinolide on thedegree of inclination of etiolated leaf lamina in wild type, d61-1, andd61-2 plants.

[0094] The highest response of the leaf from wild type (panel A) andreduced response in mutant plants d61-1 (panel B) and d61-2 (panel C)are shown.

[0095]FIG. 7 is a drawing which shows amounts of brassinosteroids inwild type and d61-2 rice plants, and biosynthetic precursors thereof.

[0096] The amounts (ng/g fresh weight) of each compound in mutant(upper) and wild type (lower) plants are shown. ND indicates notdetected.

[0097]FIG. 8 is a photograph which shows de-etiolation phenotype of thed61 in the dark.

[0098] (A) Wild type

[0099] Left: seedlings grown for two weeks in the dark

[0100] Right: seedlings grown for two weeks in the light

[0101] The internode elongation in wild type (A) and two gibberellindeficient rice mutants, d18 (D) and d35 (E), are indicated in the dark.

[0102] (B) d61-1 mutant

[0103] Left: seedlings grown for two weeks in the dark

[0104] Right: seedlings grown for two weeks in the light

[0105] The white arrows indicate internode elongations in right of eachpanel, the dark condition. No elongation was observed in the light (leftof each panel).

[0106] (C) d61-2 mutant

[0107] Left: seedlings grown for two weeks in the dark

[0108] Right: seedlings grown for two weeks in the light

[0109] No internode elongation in d61 mutant, d61-1 (B) and d61-2 (C),is observed even in the dark. The present inventors stripped the leafsheath of plants grown in the dark.

[0110] (D) d18 mutant

[0111] Left: seedlings grown for two weeks in the dark

[0112] Right: seedlings grown for two weeks in the light

[0113] (E) d35 mutant

[0114] Left: seedlings grown for two weeks in the dark

[0115] Right: seedlings grown for two weeks in the light

[0116]FIG. 9 represents a drawing and photograph which show the stronglinkage between the d61 locus and OsBRI1.

[0117] (A) is a drawing which indicates map position of the d61 locus onthe long arm of chromosome 1.

[0118] (B) is a photograph which indicates the result of DNAhybridization analysis to test the linkage between the d61 locus andOsBRI1. The RFLP of OsBRI1 was observed between the Japonica parent T65(lane T, 12.5 kb), and the Indica parent Kasarath (lane K, 17.5 kb) whenthe genomic DNAs were digested with EcoRI. Plants with a normalphenotype (WT) were heterozygous (12.5+17.5 kb) or homozygous for theIndica allele (17.5 kb), while the plants with the mutant phenotype(mutant) were always homozygous for the Japonica allele (12.5 kb).

[0119]FIG. 10 shows comparison of the deduced amino acid sequences ofOsBRI1 and Arabidopsis BRI1. Identical residues are shaded. Theunderlined regions, *1, *2, *3, and *4, indicate: a putative signalpeptide, a leucine zipper motif, N, and C sides of a cysteine pair.

[0120]FIG. 11 is a continuation of FIG. 10.

[0121]FIG. 12 is a photograph which shows expression pattern of OsBRI1in various organs.

[0122] Total RNA (10 μg) from various organs of wild type plants wereloaded into each lane.

[0123] Organ specific expression of OsBRI1

[0124] (A) Leaf blade (lane 1), leaf sheath (lane 2), developed flower(lane 3), rachis (lane 4), shoot apex (lane 5), root (lane 6), and seed(lane 7).

[0125] Region specific expression of OsBRI1 in the developing firstinternode

[0126] (B) Node (lane 1), divisional zone (lane 2), elongation zone(lane 3), and elongated zone (lane 4) in developing internodes.

[0127] Differential expression of OsBRI1 in each elongating internode

[0128] (C) The divisional and elongation zones of the first to fourthinternodes, respectively, at the actively elongating stage for eachinternode (lanes 1-4). OsBRI1 was expressed at a high level also in theunelongated stem at the vegetative phase (lane 5).

[0129] Light-dependent and brassinolide-dependent expression of OsBRI1

[0130] (D) Rice seedlings were grown for ten days in the light (lanes 1and 2) or dark (lane 3 and 4) on agar plate in the presence (lane 2 and4) or absence (lane 1 and 3) of 1 μM brassinolide.

[0131]FIG. 13 is a photograph which shows phenotype of the transgenicrice plants expressing the antisense strand of OsBRI1.

[0132] (A) Dwarf phenotype of OsBRI1 antisense plants with intermediate(centre) and severe phenotypes (right) compared to a wild type plant(left).

[0133] (B) Close up view of a transgenic plant with severe phenotype.

[0134] Bar=5 cm.

[0135] (C) Naked culm internodes of a transgenic plant. From left toright, wild type plant with normal elongation pattern of internodes andtransgenic plants with the dm, dm-d6, and d6 phenotypes are shown,respectively.

[0136] (D) Leaf morphology of wild type (left) and transgenic plantswith mild phenotype (right), showing the erect leaves in the latter.

[0137] (E) Abnormal leaf morphology of a transgenic plant with a severephenotype, showing lack of developed sheath organs. Bar=10 cm.

[0138] (F) Panicle morphology in wild type (left) and transgenic plantswith the mild (centre) and intermediate (right) phenotypes

[0139]FIG. 14 is a photograph which shows the phenotype of a transgenicplant that expresses dominant negative of OsBRI1. The transgenic plant(left) and a control plant containing vector without any inserts (right)are shown.

BEST MODE FOR CARRYING OUT THE INVENTION

[0140] The present invention is specifically illustrated below withreference to Examples, but it is not to be construed as being limitedthereto.

[0141] Rice (Oriza sativa) seeds were soaked in distilled water and madeto imbibe for 48 h at 30° C. After washing the seeds with distilledwater several times, the seeds were germinated in a dark chamber at 30°C. for 8 days. Rice plants were grown in the field or in the greenhouseat 30° C. (day) and 24° C. (night).

EXAMPLE 1 Characterization of Rice d61 Dwarf Mutants

[0142] Rice dwarf mutants d61-1 and d61-2 were obtained by treatmentwith N-methyl-N-nitrosourea (NMU), respectively (FIG. 2A).

[0143] d61 mutants carrying the weak allele specifically fail toelongate the second internode (dm-type), while those with the strongallele fail to elongate all of the internodes except the uppermost one(d6-type) (Wu, X. et al. (1999) Bread. Sci. 49, 147-153).

[0144] The culm of d61-2 is much shorter than that of d61-1. Inaddition, d61-1 shows typical dm-type pattern of internode elongation,while d61-2 shows the d6-type (FIG. 2B). Thus, they were initiallycharacterized as two independent mutants. However, crossing testdemonstrated that they are alleles on a single locus. This is the firstexample of rice mutants of a single locus which show different, specificpatterns of inhibition of internode elongation.

[0145] These mutants have other abnormal phenotypes as a result ofpleiotropic effect, as well as that inhibiting internode elongationspecifically. For example, the neck internode of the mutants is longerthan that of wild type plants (FIG. 2C). As the neck internode lengthshows an inverse relationship to the length of culm in these plants,wild type plant thus has the longest culm and shortest neck internode,the d61-1 mutant has intermediate length culm and neck internodes, andd61-2 has the shortest culm and the longest neck internode.

[0146] Another abnormal phenotype of these mutants is erect leaves (FIG.2D). In wild type plants, the leaf blade bends away from the verticalaxis of the leaf sheath towards the abaxial side. The leaf blade bendsaway from the leaf sheath at a specific organ, lamina joint, which isindicated by arrows in FIG. 2D. When the leaf blades and sheaths arefully elongated, cells at the adaxial side of the lamina joint start toelongate causing the leaf blade to bend away form the leaf sheath.However, the leaf blade of a mutant does not clearly bend away from theleaf sheath. In the d61-1 mutant some leaves still show slight bending(FIG. 2D, centre), but in the d61-2 mutant almost all the leaves arecompletely erect (FIG. 2D, right).

[0147] That is, the degree of lamina inclination correlates to theseverity of the dwarfism. The continuity of the severity in laminainclination suggests that the longitudinal elongation of the surfacecells on the adaxial side of lamina, which causes the laminainclination, responds continuously to the brassinosteroid signal.

[0148] The lack of bending of the mutant leaves is not caused by none orless development of the lamina joint. Indeed, even the d61-2 mutant withthe severe phenotype in lamina joint developed normally. The mutantsshowed shorter leaf sheaths than that of the wild type plants (FIG. 2E).

EXAMPLE 2 Observation of Cell Morphology in Internode

[0149] Internode elongation is caused by cell division in theintercalary meristem and cell elongation in the elongation zone(Hoshikawa, K. (1989) Stem. In: The growing rice plant. (Nobunkyo), pp.123-148). Therefore, dwarfing of the culms could be due to a defect inone or both of these processes. To distinguish between thesepossibilities, the present inventors examined sections of each internodefrom adult plants under the microscope.

[0150] Developing or developed culms at various stages were fixed in FAA(formalin: glacial acetic acid: 70% ethanol, 1:1:18), and dehydrated ina graded ethanol series. The samples were embedded in a Technovit 7100resin (Kurzer, Germany) polymerized at 45° C. and 3-5 μm sections werecut, stained with Toluidine Blue and observed under the lightmicroscope.

[0151]FIG. 3 shows the cell morphology of the upper four internodes in awild type plant and the d61-2 mutant. In the wild type plant, cells inall internodes were longitudinally elongated and well organized withlongitudinal files (FIG. 3A, 3B, 3C, and 3D). Similar longitudinal cellfiles were also seen in the first internodes of the mutant plants,although the cells were a little shorter than those in the wild typeplant (FIG. 3E). In the non-elongated internodes of the mutant, such asthe second and third internodes, the arrangement of cells wasdisorganized with no organized cell files apparent (FIG. 3F and 3G).Disorganization of the internodal cells indicates that the intercalarymeristems of the mutants, which normally give rise to the longitudinalfiles of elongated cells, are not developed in the non-elongatedinternodes. In the fourth internode, organized cell files were presentbut the cells were much shorter than that of the wild type plant(compare FIG. 3D and 3H). This suggests that intercalary meristems diddevelop in these internodes but the cells failed to elongate.

EXAMPLE 3 Observation of the Arrangement of Microtubules

[0152] It was described in detail that cell elongation depends on theorientation of microfibrils (Ledbetter, M. C. and Porter, K. R. (1963)J. Cell Biol. 19, 239-250; Green, P. B. (1962) Science. 138, 1404).Therefore, the present inventors examined the arrangement ofmicrotubules in the internodal cells of wild type and d61-2 mutantplants by immunofluorescence microscopy.

[0153] Microtubules in internodal parenchyma tissue were strainedimmunofluorescently. More specifically, internodal parenchyma tissue wasprefixed for 45 min at room temperature in 3.7% (w/v) paraformaldehydein microtubule-stabilizing buffer (0.1 M piperazine-diethanolsulphonicacid, 1 mM MgCl₂, 5 mM ethyleneglycol-bis(3-aminomethylether-N,N,N′,N′-tetraacetic acid, 0.2% (v/v) Triton X-100,1% (w/v) glycerol, pH 6.9). Longitudinal sections were cut with a freshrazor blade, collected in the fixation solution, treated for 40 mintherein, and washed in the fixation solution without paraformaldehyde.The sections were then incubated with a rabbit anti-α-tubulin monoclonalantibody, diluted 1:500 in phosphate-buffered saline containing 0.1%(v/v) Triton X-100, for 1 h at 37° C. The sections were washed threetimes in the buffer without anti-serum and then incubated overnight at4° C. with fluorescein-isothiocyanate-labeled mouse anti-rabbit IgGantibody diluted 1:50 in phosphate-buffered saline containing 0.1% (v/v)Triton X-100. After three washes in the same buffer, they were mountedin antifading solution (Fluoro Guard Antifade reagent, Bio-Rad) andobserved under a fluorescence microscope.

[0154] As a result, in wild type plants, the microtubules in cells ofelongating internodes were arranged in an orderly manner at right anglesto the direction of elongation (FIG. 4A). However, in the d61-2 mutant,the microtubules in cells of the first elongating internode werearranged in different directions in each cell, apparently at random(FIG. 4B). In addition, the microtubules in the mutant appeared to bethinner and less distinct relative to those of the wild type plant.

[0155] Moreover, the present inventors were unable to observe anyorganized microtubule arrangement in cells from non-elongated internodesof the mutant plants.

[0156] Taken together with the results in Example 2 show thatnon-elongated internodes in the mutants fail to develop an intercalarymeristem and the cells lack an organized arrangement of microtubules. Inthose internodes that do elongate in the mutants, although less than inthe wild type plants, an intercalary meristem does develop but the cellslack a well-organized microtubule arrangement.

EXAMPLE 4 Test of the Sensitivity Against Brassinosteroids

[0157] The dwarf phenotype and erect leaves of the d61 mutant suggeststhe possibility that the D61 gene product could be involved in eitherthe biosynthesis or signal transduction of brassinosteroids. Thus, thepresent inventors carried out the following experiments.

[0158] First, the present inventors attempted to restore the dwarfism ofthe d61 mutants, but could not be achieved it by the application ofbrassinolide.

[0159] Next, seeds of the wild type and mutant plants were germinated onagar plates with or without 1 μM brassinolide. The characteristics ofwhole seedlings were observed 1 day after germination.

[0160] The result showed that, when the wild-type plants were germinatedon the plates with brassinolide, the coleoptiles of wild type plantselongated abnormally resulting in a twisted shape and the foliage leavesgrew poorly and did not break through the coleoptile (FIG. 5).Furthermore, root elongation was inhibited and thus the roots were notstraight but developed in a wavy form. The wild type plants grewnormally in the absence of brassinolide, with coleoptile elongationstopping at an early stage of germination and then foliage leaveselongating to break out of the coleoptile. Roots developed normally anddid not have any wavy form (FIG. 5). In contrast to the wild typeplants, the mutants showed normal growth patterns, as well as that ofthe wild type plants on plates without brassinosteroids, even in thepresence of brassinosteroids. These results suggest that the mutantplants are less sensitive to brassinolide than the wild type plants.

[0161] The present inventors further tested the sensitivity of themutants to brassinolide using a more quantitative method.

[0162] The degree of bending between the leaf blade and leaf sheath inrice is well known to be sensitive to the concentration of brassinolideor its related compounds. This unusual character of rice leaf is thebasis for a quantitative bioassay for brassinosteroids, known as thelamina joint test (Wada, Ketal. (1981) Plant and Cell Physiol. 22,323-325), even though the biological function of endogenousbrassinosteroids in monocotyledonous plants including rice remainsunknown. If the mutants are less sensitive to brassinosteroids, thedegree of bending between the leaf blade and sheath of the mutant plantswill be less than that of the wild type plants.

[0163] Lamina joints of first leave of wild type T65 plant (thebackground strain of the mutants), d61-1 mutant, and d61-2 mutant weretested with 10⁻³ and 10⁻² μg/ml of brassinosteroids, respectively.

[0164] As a result, the leaf blade of wild type T65 plants was bentalmost at right angles to the axis of the leaf sheath in the absence ofexogenous brassinolide becoming even more bent in the presence ofincreasing concentrations of brassinolide (FIG. 6A). When the mutantswere used for the test, the degree of bending increased with higherconcentrations of brassinolide as in the wild type plants. However, theabsolute degree of bending of the leaves from the mutants was much lessthan that of the wild type plants under the same conditions (FIG. 6B andC). This was particularly evident with the d61-2 mutant in which controlleaves were almost straight and leaves treated with 10⁻² μg/mlbrassinolide were bent at less than a right angle to the leaf sheathaxis. The results of the lamina joint test confirm that the sensitivityof the mutants to brassinosteroids is less than that of the wild typeplants.

EXAMPLE 5 Quantitative Analysis of Brassinosteroids in the d61-2 Mutant

[0165] Example 4 demonstrates that the mutants have reduced sensitivityto brassinosteroids. That is, mutants may synthesize higherconcentrations of brassinosteroids to compensate for the reduction.Thus, the present inventors measured the concentration ofbrassinosteroids in both the d61-2 mutant and wild type plants usingGC-SIM with internal standards.

[0166] Wild type and d61-2 plants were grown in a greenhouse with a16-hrs day and 8-hrs night. Shoots from 2-month old plants wereharvested and then immediately lyophilized. Lyophilized shoots (50 gfresh weight equivalent) were extracted twice with 250 ml of MeOH-CHCl₃(4:1 [v/v]), and deuterium-labeled internal standards (1 ng/g freshweight) were added thereto. The extract was partitioned between CHCl₃and H₂O after evaporation of the solvent in vacuo. The CHCl₃-solublefraction was subjected to silica gel chromatography (Wako-gel C-300;Wako; 15 g). The column was sequentially eluted with 150 ml each ofCHCl₃ containing 2% methanol and CHCl₃ containing 7% methanol. Eachfraction was purified by Sephadex LH-20 column chromatography, where thecolumn volume was 200 ml and the column was eluted with methanol-CHCl₃(4:1 [v/v]). The fractions eluting from 0.6 to 0.8 (V_(e)/V_(t)) werecollected as brassinosteroid fractions. After pre-purification on an ODScartridge column (10×50 mm [internal diameter×column length]) in MeOH,the eluates derived from 7% MeOH fractions were subjected to ODS-HPLC ata flow rate of 8 ml/min with 65% acetonitrile as the solvent. In HPLCpurification, the 7% methanol eluate was resolved into castasterone(retention time from 10 to 15 min), typhasterol (25 to 30 min),6-deoxocastasterone (40 to 45 min) fractions, and the 2% methanol eluategave a 6-deoxotyphasterol fraction (55 to 60 min). Each fraction wasderivatized and analyzed by GC-SIM. The endogenous levels ofbrassinosteroids were calculated from the ratio of the peak areas of theprominent ions from the endogenous brassinosteroids and the internalstandard.

[0167] As a result, brassinosteroid was not detected in shoots fromeither the mutant or wild type plants, suggesting that brassinolide is aminor component of the total brassinosteroids pool. However, all of theother brassinosteroids were detected in both plant types, with theexception of teasterone which was not found in the wild type plants. Thecontents of all of the brassinosteroid compounds detected were greaterin the mutant plants (FIG. 7). In particular, castasterone was fourtimes higher in the mutant than in the wild plant. These results supportthe hypothesis that the mutants have no sensitivity to brassinosteroids.

EXAMPLE 6 De-Etiolation Phenotype of the d61 Mutants

[0168] Reduction of hypocotyl elongation and emergence of opening of thecotyledons and primary leaves in complete darkness are reported inArabidopsis mutants with deficiencies in brassinosteroid biosynthesis orbrassinosteroid signaling when grown in the dark (Kauschmann, A et al.(1996) Plant J. 9, 701-703; Szekeres, M. et al. (1996) Cell. 85,171-182).

[0169] This de-etiolated (DET) or constitutive photomorphogenesis (COP)phenotype in darkness is a common feature of Arabidopsisbrassinosteroids-related mutants. A similar DET or COP phenotype is alsoobserved in a tomato dwarf (d) mutant that shows a short hypocotyl, lackof apical hook, and expansion of cotyledons (Bishop, G. J. et al. (1996)Plant Cell. 8, 959-969). In contrast, a pea brassinosteroids-defectivemutant, 1 kb, does not show such a DET phenotype (Nomura, T. et al.(1997) Plant Physiol. 93, 572-577). Mutants were grown in the dark todetermine whether such DET or COP phenotypes were also found inmonocotyledonous plants and whether the d61 rice mutants showedcharacteristics of skotomorphogenesis.

[0170] As a result, when wild type plants were germinated in the dark,they showed unusual elongation of the mesocotyl and internodes comparedto light-grown seedlings (FIG. 8A). Such elongation of the mesocotyl andinternodes did not occur in the mutants even in the dark (FIG. 8B and8C). This failure of the mesocotyl and internodes to elongate in thedark is not a common characteristic of rice dwarf mutants. For example,two other dwarf mutants, d18 and d35, which are deficient in gibberellinbiosynthesis, showed elongated mesocotyls and internodes when grown inthe dark (FIG. 8D and 8E, respectively).

[0171] Thus, it is conceivable that the reduced elongation of themesocotyl and internodes are specific feature of the d61 mutants andthat the d61 mutants have de-etiolated phenotype. In addition, it isalso indicated that de-etiolation due to defects in brassinosteroidsignal is common characteristic in both dicotyledonous andmonocotyledonous plants. That is, it is conceivable that brassinosteroidsignals are important for skotomorphogenesis in both dicotyledonous andmonocotyledonous plants.

EXAMPLE 7 Mapping and Linkage Analysis of D61 Locus

[0172] For mapping of the D61 locus, the present inventors crossed thed61-2 mutant with an Indica rice cultivar, Kasarath (Oriza sativa L. cv.Kasazath). The linkage analysis between the mutant phenotype andrestriction fragment polymorphism (RFLP) markers released from the RiceGenome Project (Tsukuba, Japan) revealed that the D61 locus maps to thelong arm of chromosome 1, with tight linkage to the RFLP marker, C1370(FIG. 9A).

[0173] As d61 could be characterized as a mutant with no or reducedsensitivity to brassinosteroid, the present inventors also tested thelinkage between the mutant phenotype and a rice gene that is homologousto the Arabidopsis BRI1 gene.

[0174] The Arabidopsis BRI1 gene was isolated as the only gene that isinvolved in brassinosteroid signal transduction (Li, J. and Chory, J.(1997) Cell. 90, 929-938). Furthermore, the present inventors carriedout a BLAST search to identify one rice EST clone, S1676, with highhomology to the Arabidopsis BRI1 gene (Li, J. and Chory, J. (1997) Cell.90, 929-938).

[0175] Rice genomic DNA was isolated from leaf tissue using an ISOPLANTDNA isolation kit (Nippon GENE Co., Japan). One μg of the genomic DNAwas digested with appropriate restriction enzymes and transferred ontoHybond N⁺ membranes (Amersham) under alkaline conditions. The membranewas probed using the partial cDNA fragment (corresponding to the regionfrom Ser 740 to Asp 1116 in the kinase domain), which specificallyhybridized with the genomic DNA fragment encoding OsBRI1. All of thesteps were carried out according to the method described by Church andGilbert (1984) PNAS. 81, 1991-1995, except that membranes werehybridized at high stringency (68° C.).

[0176] An RFLP between the Japonica (12.5 kb) and Indica (17.5 kb) ricewas observed when genomic DNAs were digested with EcoRI and probed withthe cDNA clone. All F2 plants with the mutant phenotype were homozygousfor the Japonica allele (12.5 kb), whereas the F2 plants with the wildtype phenotype were either homozygous for the Indica allele (17.5 kb) orheterozygous with both the Japonica and Indica alleles (12.5+17.5 kb,FIG. 9B). This result demonstrates that the D61 locus is closely linkedto the position of the rice gene that is homologous to the ArabidopsisBRI1.

EXAMPLE 8 Identification of the d61 Gene

[0177] The above linkage analysis strongly suggested that the d61mutation is caused by loss of function of the rice homologue of theArabidopsis BRI1 gene. To test this possibility, the present inventorsscreened rice genomic DNA library with probes and isolated the entirelength. of the rice BRI1 homologous gene (OsBRI1, Oryza sativa BRI1).Hybridization in this screening was performed as described in Church andGilbert (1984) except that membranes were hybridized at higherstringency (68° C.). Sequencing was carried out according to the samemethod described by Church and Gilbert.

[0178] The structure of the OsBRI1 gene is quite similar to theArabidopsis BRI1 gene in its entire length (FIG. 10 and FIG. 11). Thepredicted OsBRI1 polypeptide contains several domains that are alsopresent in the BRI1, and the functions of which were discussed (Li, J.and Chory, J. (1997) Cell. 90, 929-938). These domains consist of aputative signal peptide, two conservatively spaced cysteine pairs, aleucine-rich repeat domain, a transmembrane domain, and a kinase domain.The N-terminus of the predicted OsBRI1 polypeptide has a hydrophobicsegment which is predicted to act as a signal peptide to transport theprotein to the plasma membrane. In the BRI1 protein, Li and Chorypredicted a potential 4-hepted amphipathic leucine zipper motiffollowing the signal peptide (Li, J. and Chory, J. (1997) Cell. 90,929-938), but OsBRI1 does not have such a typical leucine zipper motifin the corresponding region.

[0179] A putative extracellular domain (from Met¹ to Leu⁶⁷⁰), consistingof 22 tandem copies of a leucine-rich repeat (LRR) of about 24-aminoacids with 12 potential N-glycosylation sites (Asn-X-Ser/Thr), isflanked by pairs of conservatively spaced cysteines. The LRR has beenimplicated to function in protein-protein interactions (Kobe, B. andDeisenhofer, J. (1994) Trends Biochem. Science. 19, 415-421).

[0180] In comparison to the BRI1 sequence, OsBRI1 lacks three LRRdomains corresponding to the third to fifth repeat of the ArabidopsisBRI1. The two LRRs before this deletion are less conserved except forthe consensus residues found between other LRR proteins, but the LRRs ofboth proteins are well conserved after the deletion in both the LRRconsensus residues and non-conservative amino acids. An unusual featureof the LRR region of BRI1 is the presence of a 70-amino acid islandbetween the 21st and 22nd LRR (Li, J. and Chory, J. (1997) Cell. 90,929-938). A highly similar feature is also present in OsBRI1 with thesame number of amino acids between the 18th and 19th LRRs correspondingto the site of island in BRI1. This unusual amino acid island in LRRregion must be important for functions thereof, because exchange of anamino acid residue in this island resulted in the loss of function ofBRI1 (Li, J. and Chory, J. (1997) Cell. 90, 929-938) This motif wasthought to be important for direct interaction with brassinosteroids orfor maintaining the structure of the brassinosteroids-binding domain(Li, J. and Chory, J. (1997) Cell. 90, 929-938).

[0181] The protein kinase domain of OsBRI1 has all eleven conservedsubdomains of eukaryotic protein kinases, retaining the invariant aminoacid residues in their proper positions (Hanks, S. K. and Quinn, A. M.(1991) Meth. Enzymol. 200, 38-62). The protein kinase domain of OsBRI1is highly related to that of BRI1 (44%) over the entire region. It isalso related to the kinase domains of other receptor-like proteinkinases in higher plants such as ERECTA (Torii, K. U. et al. (1996)Plant Cell. 8, 735-746), CLV1 (Clark, S. E. et al. (1997) Cell. 3,575-585), and RLK5 (Walker, J. C. (1993) Plant J. 3, 451-456) fromArabidopsis, and Xa2l from rice (Song et al., 1995). The highlyconserved structure of OsBRI1 and these receptor-like protein kinases,especially in subdomains VIb and VIII, suggests that OsBRI1 is aserine/threonine kinase rather than a tyrosine kinase (Hanks, S. K. andQuinn, A. M. (1991) Meth. Enzymol. 200, 38-62).

EXAMPLE 9 Sequencing of OsBRI1 Gene in d61-1 and d61-2 Mutants

[0182] The present inventors also determined the entire sequences of theOsBRI1 gene in the d61-1 and d61-2 mutants, and compared them to that ofthe wild type plant. The present inventors identified a singlenucleotide substitution in each mutant allele at different sites (Table1). The genomic mutation in d61-1 resulted in exchange from threonine toisoleucine at residue 989 in subdomain IX of the kinase domain which isconserved between OsBRI1 and BRI1. The genomic mutation in d61-2 changedvaline to methionine at residue 491 in the 17th LRR, just before theunusual 70-amino acid interrupting region. These mutations in the OsBRI1genes from the d61 mutants provide strong evidence that OsBRI1 encodesthe D61 locus. TABLE 1 Alleles Characteristics of mutation Position ofcoding sequence d61-1 C → T Thr → Ile (989) d61-2 G → A Val → Met (491)

EXAMPLE 10 Molecular Complementation Analysis of the d61 Mutation by theIntroduction of OsBRI1 Gene

[0183] To confirm that OsBRI1 corresponds to the d61 locus, the presentinventors carried out complementation analysis of the d61-1 mutant byintroduction of the wild-type OsBRI1 gene.

[0184] More specifically, to confirm complementarity of d61 phenotypedue to introduction of a genomic OsBRI1 clone including its 5′ and 3′flanking regions, a 10.5-kb restriction fragment including the entirecoding region was cloned into the XbaI-SmaI sites of the hygromycinresistance binary vector pBI101-Hm3 (Sato, Y. et al. (1999) EMBO J. 18,992-1002). pBI-cont was used as a control vector. The present inventorsperformed rice tissue culture and Agrobacterium tumefaciens mediatedtransformation.

[0185] Transformation of d61 with a control vector that carries no ricegenomic DNA had no apparent effect on the culm length or the structureof leaves. However, when a 10.5 kb DNA fragment containing the entirewild-type OsBRI1 gene was introduced, the normal phenotype was recoveredin almost plants that were resistant to hygromycin. This result confirmsthat the d61 mutant phenotype is caused by the loss-of-function mutationin the OsBRI1 gene.

EXAMPLE 11 RNA Hybridization Analysis of OsBRI1

[0186] Nothing is known about the function of endogenousbrassinosteroids in monocotyledonous plants. Therefore, the presentinventors tested the expression pattern of the OsBRI1 gene in variousrice organs by RNA hybridization analysis.

[0187] RNA was isolated from various rice tissues as described inliterature (Chomczynski, P. and Sacchi, N.: Anal. Biochem. (1987)162:156). Ten μg of total RNA were electrophoresed in a 1% agarose gel,then transferred to a Hybond N⁺ membrane (Amersham), and analyzed by RNAgel blot hybridization. The present inventors used a partial cDNAfragment (Ser⁷⁴⁰ to Asp¹¹¹⁶ corresponding to the kinase domain), as aprobe which specifically hybridized to the genomic DNA fragment encodingOsBRI1. DNA hybridization analysis was performed with the same probe andhybridization conditions as described above.

[0188] As a result, a single, strongly-hybridizing band was detected inRNA from vegetative shoot apices (FIG. 12A). The size of the band wasapproximately 3.5 kb, which is almost the same size as the longest cDNAclone. More weakly-hybridizing bands of the same size were also observedin RNA from flowers, rachis, roots, and expanded leaf sheaths, while noor very faint bands were observed in RNA from expanded leaf blades.Thus, the expression of OsBRI1 varies markedly between organs suggestingthat the sensitivity to brassinosteroids also differs among theseorgans.

[0189] The present inventors also examined the expression of OsBRI1 inelongating culms (FIG. 12B). Elongating culms were divided into fourparts: the node and the division, elongation, and elongated zones of theinternode. As a result, the most strongly hybridizing band was found inRNA from the division zone. RNA from the elongation zone also gave astrong signal. RNA from the node gave only a weak signal, whilst thatfrom the elongated internode gave no signal at all. These resultsindicate that the elongating culm has different sensitivities tobrassinosteroids partially, with the most sensitive parts being thedivision and elongation zones where cells are actively dividing andelongating.

[0190] The present inventors further examined the expression of OsBRI1in the elongation zones of the upper four internodes at the stage wheneach internode was actively elongating. A strongly hybridizing band wasfound in RNA from the elongation zone of the uppermost (first) and thelowest (fourth) internodes, while relatively weak bands were seen withthe second and third internodes (FIG. 12C). This result indicates thatthe internodes differ in their sensitivity to brassinosteroids, with thesecond and third internodes being the least sensitive.

[0191] It was observed that the internodes differ in their sensitivityto brassinosteroids. It suggests that the uppermost and fourthinternodes have higher sensitivity to brassinosteroids than the secondand third internodes, if the amount of OsBRI1 is a limiting factor inbrassinosteroid signal transduction. This idea is supported by themutant allele with the intermediate, dm-d6 type phenotype. These plantsshow specific reduction of the second and third internodes while theuppermost and fourth internodes are elongated. This is consistent withthe second and third internodes, with lower expression level of OsBRI1,having lower sensitivity to brassinosteroids such that they are unableto respond to the brassinosteroid signal and elongate. Presumably, thehigher OsBRI1 expression level in the uppermost and fourth internodesdoes allow these internodes to respond to the brassinosteroid signal andelongate. The higher expression level of OsBRI1 in the uppermost andfourth internodes can explain the unusual internode elongation patternof the dm-d6 type, but it cannot explain the occurrence of the d6 or dmtype. The d6 type, in which all of the internodes except the uppermostare reduced, could indicate that the uppermost internode is exposed tohigher levels of brassinosteroids than the fourth internode. The timingof the elongation of the uppermost internode corresponds with thedevelopment of anthers in the flowers, and high level ofbrassinosteroids have been observed in these organs in many plants(Grove, M. et al. (1979) Nature, 281, 216-217; Plattner, D. et al.(1986) J. Natural Products. 49, 540-545; Ikekawa, N. et al. (1988) Chem.Pharm. Bull. 36, 405-407; Takatuto, S. et al. (1989b) Agric. Biol. Chem.53, 2177-2180; Suzuki, Y. et al. (1986) Agric. Biol. Chem. 50,3133-3138; Gamoh, K. et al. (1990) Anal. Chim. Acta. 228, 101-105). Itappears that high levels of brassinosteroids move down from the anthersto lower organs, such as, the uppermost internode and induce internodeelongation and that the fourth internode completes its elongation beforeflower development and does not receive the high level brassinosteroidsignal from the flowers at the time of its active elongation. Thebrassinosteroid level and the sensitivity to brassinosteroids of OsBRI1cannot explain the specific retardation of the second internode observedin the dm-type mutants. Therefore, some other factor(s) must beinvolved. It seems likely that elongation of the second internode isregulated by several factors, since there are several independent dwarfmutants with the dm-phenotype including d1, d2, d11, and d61.

[0192] Very recently, the D1 gene was isolated and found to encode aprotein with a similar structure to the α subunit (G-α) of a G protein(Fujisawa, Y. et al. (1999) Proc. Natl. Acad. Sci. USA. 96,7575-7580;Ashikari, M. et al. (1999) Proc. Natl. Acad. Sci. USA. 96, 10284-10289)The D1 G-α like protein is now thought to be involved in the gibberellin(GA) signal transduction pathway, since the d1 mutant alleles show lowor no sensitivity to active GA. It is interesting that theloss-of-function mutants of the brassinosteroid signal-related protein,OsBRI1, and that of the GA signal-related protein, Gα, show the samephenotype, i.e., specific retardation of the second internode. Thus, inthe induction of elongation in the second internode, there could be aspecific mechanism common to brassinosteroids and GA signal transductionin the second internode.

[0193] Interestingly, high level expression of OsBRI1 was also seen inthe stem at the vegetative stage, in which the internodes do notelongate, showing that high level expression of OsBRI1 in the culm doesnot necessarily coincide with internode elongation.

EXAMPLE 12 Effect of Exogenous Brassinolide and Light on the Level ofOsBRI1 mRNA

[0194] The present inventors also tested the effects of exogenouslyapplied brassinolide and light on the level of OsBRI1 mRNA. Germinatingseeds were placed on 0.9% agar plates with or without 1 μM brassinolideand grown for six days in the light or dark.

[0195] As a result, on plates without brassinosteroids, the expressionlevel of OsBRI1 in dark grown seedlings was higher than in light grownseedlings (FIG. 12D).

[0196] This suggests that the dark-grown rice seedlings have a highersensitivity to brassinosteroids than the light-grown plants (Worley, J.F. and Mitchell, J. W. (1971) J. Amer. Soc. Hort. Sci. 96, 270-273).High sensitivity to brassinosteroids in dark-grown plants will due tothe elongation of internode cells in situations where the cells inlight-grown plants do not respond to brassinosteroids.

[0197] Furthermore, on plates with brassinosteroids, both of the light-and the dark-grown rice seedlings had a reduced level of OsBRI1expression.

[0198] In contrast to rice, the level of BRI1 expression in Arabidopsisis little changed between dark- and light-grown seedlings (Li, J. andChory, J. (1997) Cell. 90, 929-938). The reason for this differencebetween the expression patterns of the rice OsBRI1 and that of theArabidopsis BRI1 is not known. However, the difference could be relatedto the difference in photoresponse mechanisms that rice is short-dayplant, while Arabidopsis is long-day plant.

EXAMPLE 13 Phenotypic Analysis of Transgenic Plants Expressing theAntisense Strand of OsBRI1

[0199] The above phenotypic analyses of the d61 mutants and the singlenucleotide exchange in the OsBRI1 genes in each mutant suggest that theymight not be null alleles and could retain some partial function. Toinvestigate further the function of brassinosteroids in rice, thepresent inventors attempted to generate other mutants with more severephenotypes by overexpression of the antisense strand of the OsBRI1transcript under the control of the rice Actin1 gene promoter (Zang, W.et al. (1991) Plant Cell. 3, 1155-1165).

[0200] For constructing Actin1 promoter::antisense OsBRI1, apromoter-terminator cassette (pBIAct1nos) containing the Act1 promoter(Zhang, W. et al. (1991) Plant Cell. 3, 1155-1165) and NOS terminatorwas constructed by substitution of the Act1 promoter for the 35Spromoter in the hygromycin resistance binary vector, pBI35Snos (Sato, Y.et al. (1999) EMBO J. 18, 992-1002), which contains the 35S promoter andNOS terminator, between the HindIII and XbaI sites. The cDNA cloneencoding entire OsBRI1 coding region was introduced between the XbaI andSmaI sites of pBIAct1nos. Vector pBI-cont, containing no insert was usedas a control vector.

[0201] For reduction of OsBRI1 expression, the OsBRI1-antisense cDNAwere introduced into the rice cultivar Nipponbare. The present inventorsperformed rice tissue culture and Agrobacterium tumefaciens mediatedtransformation.

[0202] Almost all of the resulting transgenic plants (90% or more, 18out of 20) produced erect leaves during the early stages of seedlinggrowth (FIG. 13D). All of the transformants (20 out of 20) showed adwarfed phenotype of varying severity (FIG. 13A). In the plants with theweakest phenotype, the length of each internode was partially anduniformly reduced resulting in an elongation pattern similar to that ofthe wild plant (FIG. 13C) (dn-type mutants). Plants with intermediatephenotypes had the typical internode elongation patterns of dm-type(specific reduction of the second internode, FIG. 13C) or d6-type(specific reduction of the second to fourth internodes, FIG. 13C)mutants or a mixed dm- and d6-type phenotype (specific reduction of thesecond and third internodes, FIG. 13C). Plants with the severe phenotypeonly formed abnormal leaves without developed sheath organs and theinternodes did not elongate (FIG. 13E). These kinds of plants were lessthan 15-cm high, even after cultivated for one year or more, and did notproduce seeds (FIG. 13B). The other phenotypes were inherited insubsequent generations and cosegregated with hygromycin resistance. Thecosegregation between the abnormal phenotypes and hygromycin resistance,and the similarity between the intermediate phenotype of the antisenseplants and the d61 mutants demonstrate that the antisense strand acts tosuppress the function of OsBRI1 in the transgenic plants.

EXAMPLE 14 Phenotype of Transgenic Plants Expressing Dominant Negativeof OsBRI1

[0203] Transgenic rice containing the kinase region of the OsBRI1 geneunder control of the rice actin 1 gene promoter (Zhang, W. et al.,(1991) Plant Cell, 3: 1155-1165) was produced to analyze the function ofthe rice brassinosteroid receptor. That is, the plasmid was constructedas follows so that only the carboxy terminal kinase domain ofbrassinosteroid receptor would be expressed without the amino terminusregion from the first methionine to 738th glycine. The present inventorsused a pair of the primer,5′-GGCTCTAGACAGCCATGGCGAGCAAGCGGCGGAGGCTG-3′/SEQ ID NO: 4 (5′-primer:which includes TCTAGA as XhoI site, CAGCC added to increase translationefficiency, ATG as the-initiation codon, an additional GCG encodingalanine, and AGC encoding 739th serine, following further nucleotidesencoding amino acids after 740th residue of the wild type sequence,lysine, arginine, arginine, and leucine) and5′-AGATCTACTCCTATAGGTA-3′/SEQ ID NO: 5 (3′-primer: which includes AGATCTas XbaI site and following 3′-untranslated region). These two primerswere used to amplify the kinase region of the brassinosteroid receptor.The amplified fragment was then digested with XbaI and inserted into apBI vector between XbaI-SmaI sites. The vector pBI-cont which does notcontain the insert was used as a control.

[0204] The rice cultivar Nipponbare was used to produce transgenicplants which expresses kinase domain to control OsBRI1 expression. Ricetissue culture and Agrobacterium tumefaciens mediated transformationwere performed.

[0205] As a result, most of the transgenic plants (90% or more: 25/27individuals) formed erect leaves at an early stage of seeding growth(FIG. 14). The length between the internodes was partially and equallyshortened. The phenotype was inherited to the progeny by co-segregationwith hygromycin resistance activity. The co-segregation between theabnormal phenotype and hygromycin resistance and similarities betweenthe dominant negative plants and the d61 mutant intermediate phenotypeindicate that the partial cDNA for the kinase portion has activity inthe transgenic plant and suppresses OsBRI1 function.

Industrial Applicability

[0206] The present invention provides a gene and a protein whichfunctions to increase rice brassinosteroid sensitivity. This gene isinvolved in elongation of plant internode cells and inclination ofleaves. Therefore, it is possible to produce phenotypically modifiedplants by controlling this gene. For example, by suppressing theexpression of the gene of the present invention, dwarf plants, which areresistant to lodging and which enables planting a higher number ofindividuals per unit area, can be produced, which is significant in theproduction of crop products. It is also possible to produce ornamentalplants having new aesthetic value by dwarfism of height or culm lengthof said plants via suppression of expression of DNA of the presentinvention. On the other hand, brassinosteroid sensitivity of the plantcan be increased by introducing and expressing the DNA of the presentinvention in plants, resulting in increase of height of the plant andyield of the whole plant. This is useful especially in increasing yieldof plants for animal feed.

1 6 1 3710 DNA Oryza sativa misc_feature (1)..(3710) OsBRI1 cDNA 1 atggat tcc ttg tgg gca gcg ata gcg gca ctg ttt gtg gcg gcg gcg 48 Met AspSer Leu Trp Ala Ala Ile Ala Ala Leu Phe Val Ala Ala Ala 1 5 10 15 gtggtg gtg agg ggg gcg gcg gcg gcc gac gac gcc cag ctg ctc gag 96 Val ValVal Arg Gly Ala Ala Ala Ala Asp Asp Ala Gln Leu Leu Glu 20 25 30 gag ttcagg cag gcg gtg ccg aac cag gcg gcg ctc aag ggg tgg agc 144 Glu Phe ArgGln Ala Val Pro Asn Gln Ala Ala Leu Lys Gly Trp Ser 35 40 45 ggc ggc gacggc gcg tgc agg ttc ccg ggg gcc ggg tgc cgg aac ggg 192 Gly Gly Asp GlyAla Cys Arg Phe Pro Gly Ala Gly Cys Arg Asn Gly 50 55 60 agg ctc acg tcgctg tcg ctc gcc ggc gtg ccg ctc aat gcc gag ttc 240 Arg Leu Thr Ser LeuSer Leu Ala Gly Val Pro Leu Asn Ala Glu Phe 65 70 75 80 cgc gcc gtc gcggcc acc ctg ctg cag ctc ggc agc gtc gag gtg ctg 288 Arg Ala Val Ala AlaThr Leu Leu Gln Leu Gly Ser Val Glu Val Leu 85 90 95 agc ctc cgc ggc gccaac gtc agc ggc gcg ctc tcg gcg gct ggc ggc 336 Ser Leu Arg Gly Ala AsnVal Ser Gly Ala Leu Ser Ala Ala Gly Gly 100 105 110 gcg agg tgc ggg agcaag ctg cag gcg ctc gat ttg tcc ggg aat gcc 384 Ala Arg Cys Gly Ser LysLeu Gln Ala Leu Asp Leu Ser Gly Asn Ala 115 120 125 gcg ctc cgg ggc tccgtc gcc gac gtg gcg gcc ctg gcc agc gcc tgc 432 Ala Leu Arg Gly Ser ValAla Asp Val Ala Ala Leu Ala Ser Ala Cys 130 135 140 ggc ggc ctc aag acgctg aat ctc tcc ggc gat gcg gtt ggt gcg gcg 480 Gly Gly Leu Lys Thr LeuAsn Leu Ser Gly Asp Ala Val Gly Ala Ala 145 150 155 160 aag gtc ggt ggcggt ggt ggc ccg ggc ttt gcc ggg ctg gac tcg ctt 528 Lys Val Gly Gly GlyGly Gly Pro Gly Phe Ala Gly Leu Asp Ser Leu 165 170 175 gat ttg tcc aacaac aag atc acc gac gat agc gac ctc cgg tgg atg 576 Asp Leu Ser Asn AsnLys Ile Thr Asp Asp Ser Asp Leu Arg Trp Met 180 185 190 gtg gat gcc ggagtc ggg gca gta cgg tgg ttg gac ctt gcc ctg aac 624 Val Asp Ala Gly ValGly Ala Val Arg Trp Leu Asp Leu Ala Leu Asn 195 200 205 agg atc tcc ggtgtc ccg gag ttc acc aac tgc tcc ggg ctt cag tac 672 Arg Ile Ser Gly ValPro Glu Phe Thr Asn Cys Ser Gly Leu Gln Tyr 210 215 220 ctt gac ctc tccggc aac ctc atc gtc ggt gag gtg ccc ggc ggg gca 720 Leu Asp Leu Ser GlyAsn Leu Ile Val Gly Glu Val Pro Gly Gly Ala 225 230 235 240 ctt tcc gactgc cgc ggt ctg aaa gtg ctc aac ctc tcc ttc aac cac 768 Leu Ser Asp CysArg Gly Leu Lys Val Leu Asn Leu Ser Phe Asn His 245 250 255 ctc gcc ggcgtg ttc cct ccg gac atc gcc ggc ctc acg tcg ctc aac 816 Leu Ala Gly ValPhe Pro Pro Asp Ile Ala Gly Leu Thr Ser Leu Asn 260 265 270 gcc ctc aacctc tcc aac aac aac ttc tcc ggc gag ctc ccc ggc gag 864 Ala Leu Asn LeuSer Asn Asn Asn Phe Ser Gly Glu Leu Pro Gly Glu 275 280 285 gct ttc gcaaag ctg cag cag ctt acg gcg ctc tcc ctc tcc ttc aac 912 Ala Phe Ala LysLeu Gln Gln Leu Thr Ala Leu Ser Leu Ser Phe Asn 290 295 300 cac ttc aacggc tcc atc ccg gac acc gta gcc tcg ctg ccg gag ctc 960 His Phe Asn GlySer Ile Pro Asp Thr Val Ala Ser Leu Pro Glu Leu 305 310 315 320 cag cagctc gac ctc agc tcc aac acc ttc tcc ggc acc atc ccg tcg 1008 Gln Gln LeuAsp Leu Ser Ser Asn Thr Phe Ser Gly Thr Ile Pro Ser 325 330 335 tcc ctctgc caa gat ccc aac tcc aag ctc cat ctg ctg tac ctt cag 1056 Ser Leu CysGln Asp Pro Asn Ser Lys Leu His Leu Leu Tyr Leu Gln 340 345 350 aac aactac ctc acc ggc ggc atc cca gac gcc gtc tcc aac tgc acc 1104 Asn Asn TyrLeu Thr Gly Gly Ile Pro Asp Ala Val Ser Asn Cys Thr 355 360 365 agc ctcgtc tcc ctc gac ctc agc ctc aac tac atc aat ggg tcc atc 1152 Ser Leu ValSer Leu Asp Leu Ser Leu Asn Tyr Ile Asn Gly Ser Ile 370 375 380 ccg gcatcc ctc ggc gac ctt ggc aac ctg cag gac ctc atc ctg tgg 1200 Pro Ala SerLeu Gly Asp Leu Gly Asn Leu Gln Asp Leu Ile Leu Trp 385 390 395 400 cagaac gag ctg gag ggc gag ata ccg gcg tcc ctg tcg cgc att cag 1248 Gln AsnGlu Leu Glu Gly Glu Ile Pro Ala Ser Leu Ser Arg Ile Gln 405 410 415 ggcctc gag cat ctc atc ctc gac tac aac ggg ctc acg ggt agc atc 1296 Gly LeuGlu His Leu Ile Leu Asp Tyr Asn Gly Leu Thr Gly Ser Ile 420 425 430 ccgccg gag cta gcc aag tgc acc aag ctg aac tgg att tct ttg gcg 1344 Pro ProGlu Leu Ala Lys Cys Thr Lys Leu Asn Trp Ile Ser Leu Ala 435 440 445 agcaac cgg ctg tcc ggg cca atc cct tca tgg ctt ggg aag ctc agc 1392 Ser AsnArg Leu Ser Gly Pro Ile Pro Ser Trp Leu Gly Lys Leu Ser 450 455 460 tacttg gct atc ttg aag ctc agc aac aat tcc ttc tcg ggg cct ata 1440 Tyr LeuAla Ile Leu Lys Leu Ser Asn Asn Ser Phe Ser Gly Pro Ile 465 470 475 480ccg cca gag ctc ggt gac tgc cag agc ttg gtg tgg ctg gac ctg aat 1488 ProPro Glu Leu Gly Asp Cys Gln Ser Leu Val Trp Leu Asp Leu Asn 485 490 495agc aat cag ctg aat gga tca ata ccc aaa gag ctg gcc aaa cag tct 1536 SerAsn Gln Leu Asn Gly Ser Ile Pro Lys Glu Leu Ala Lys Gln Ser 500 505 510ggg aag atg aat gtt ggc ctc ata gtt gga cgg cct tac gtt tat ctt 1584 GlyLys Met Asn Val Gly Leu Ile Val Gly Arg Pro Tyr Val Tyr Leu 515 520 525cgc aac gac gag ctg agc agc gag tgc cgt ggc aag ggg agc ttg ctg 1632 ArgAsn Asp Glu Leu Ser Ser Glu Cys Arg Gly Lys Gly Ser Leu Leu 530 535 540gag ttt acc agc atc cga cct gat gac ctc agt cgg atg ccg agc aag 1680 GluPhe Thr Ser Ile Arg Pro Asp Asp Leu Ser Arg Met Pro Ser Lys 545 550 555560 aag ctg tgc aac ttc aca aga atg tat gtg ggg agc acg gag tac acc 1728Lys Leu Cys Asn Phe Thr Arg Met Tyr Val Gly Ser Thr Glu Tyr Thr 565 570575 ttc aac aag aat ggt tcg atg ata ttt ctc gat ttg tca tat aat cag 1776Phe Asn Lys Asn Gly Ser Met Ile Phe Leu Asp Leu Ser Tyr Asn Gln 580 585590 ctg gac tcg gcg att cct ggc gag ctg ggg gac atg ttc tac ctc atg 1824Leu Asp Ser Ala Ile Pro Gly Glu Leu Gly Asp Met Phe Tyr Leu Met 595 600605 atc atg aat ctt ggg cac aac cta ctg tca ggt acc atc cca tcg cgg 1872Ile Met Asn Leu Gly His Asn Leu Leu Ser Gly Thr Ile Pro Ser Arg 610 615620 cta gca gag gcc aag aag ctt gcg gtg ctt gac ctg tcg tat aac cag 1920Leu Ala Glu Ala Lys Lys Leu Ala Val Leu Asp Leu Ser Tyr Asn Gln 625 630635 640 ttg gaa ggg cca ata ccc aac tct ttc tcg gca ctt tcc ttg tcg gag1968 Leu Glu Gly Pro Ile Pro Asn Ser Phe Ser Ala Leu Ser Leu Ser Glu 645650 655 atc aat ctg tca aat aat cag ctg aat gga aca att cca gag ctt ggt2016 Ile Asn Leu Ser Asn Asn Gln Leu Asn Gly Thr Ile Pro Glu Leu Gly 660665 670 tcc ctt gcc aca ttt ccg aag agc cag tat gag aat aac act ggt tta2064 Ser Leu Ala Thr Phe Pro Lys Ser Gln Tyr Glu Asn Asn Thr Gly Leu 675680 685 tgt ggc ttc cca ctg cca cca tgt gac cat agt tcc cca aga tct tcc2112 Cys Gly Phe Pro Leu Pro Pro Cys Asp His Ser Ser Pro Arg Ser Ser 690695 700 aat gac cac caa tcc cac cgg agg cag gca tcg atg gca agc agt atc2160 Asn Asp His Gln Ser His Arg Arg Gln Ala Ser Met Ala Ser Ser Ile 705710 715 720 gct atg gga ctg tta ttc tca ctg ttc tgt ata att gtg atc atcata 2208 Ala Met Gly Leu Leu Phe Ser Leu Phe Cys Ile Ile Val Ile Ile Ile725 730 735 gcc att ggg agc aag cgg cgg agg ctg aag aat gag gag gcg agtacc 2256 Ala Ile Gly Ser Lys Arg Arg Arg Leu Lys Asn Glu Glu Ala Ser Thr740 745 750 tct cgt gat ata tat att gat agc agg tca cat tct gca act atgaat 2304 Ser Arg Asp Ile Tyr Ile Asp Ser Arg Ser His Ser Ala Thr Met Asn755 760 765 tct gat tgg agg caa aat ctc tcc ggt aca aat ctt ctt agc atcaac 2352 Ser Asp Trp Arg Gln Asn Leu Ser Gly Thr Asn Leu Leu Ser Ile Asn770 775 780 ctg gct gca ttc gag aag cca ttg cag aat ctc acc ctg gct gatctt 2400 Leu Ala Ala Phe Glu Lys Pro Leu Gln Asn Leu Thr Leu Ala Asp Leu785 790 795 800 gtt gag gcc aca aat ggc ttc cac atc gca tgc caa att gggtct ggt 2448 Val Glu Ala Thr Asn Gly Phe His Ile Ala Cys Gln Ile Gly SerGly 805 810 815 ggg ttt ggt gat gtc tac aag gca cag ctc aag gat ggg aaggtt gtt 2496 Gly Phe Gly Asp Val Tyr Lys Ala Gln Leu Lys Asp Gly Lys ValVal 820 825 830 gca atc aag aag cta ata cat gtg agc ggg cag ggt gac cgggag ttc 2544 Ala Ile Lys Lys Leu Ile His Val Ser Gly Gln Gly Asp Arg GluPhe 835 840 845 act gca gaa atg gag acc att ggc aag atc aaa cac cgt aacctt gtt 2592 Thr Ala Glu Met Glu Thr Ile Gly Lys Ile Lys His Arg Asn LeuVal 850 855 860 cca ctt ctt ggc tat tgc aag gct ggt gag gag cgg ttg ttggtg tat 2640 Pro Leu Leu Gly Tyr Cys Lys Ala Gly Glu Glu Arg Leu Leu ValTyr 865 870 875 880 gat tac atg aag ttt ggc agc ttg gag gat gtg ttg catgac cgc aaa 2688 Asp Tyr Met Lys Phe Gly Ser Leu Glu Asp Val Leu His AspArg Lys 885 890 895 aag atc ggt aaa aag ctg aat tgg gag gca aga cgg aaaatc gct gtt 2736 Lys Ile Gly Lys Lys Leu Asn Trp Glu Ala Arg Arg Lys IleAla Val 900 905 910 gga gca gca agg ggt ttg gca ttc ctc cac cac aat tgcatt cct cac 2784 Gly Ala Ala Arg Gly Leu Ala Phe Leu His His Asn Cys IlePro His 915 920 925 atc att cac cga gac atg aag tcg agc aat gtg ctt atcgat gaa caa 2832 Ile Ile His Arg Asp Met Lys Ser Ser Asn Val Leu Ile AspGlu Gln 930 935 940 ctg gaa gca agg gta tct gat ttc ggt atg gcg agg ctgatg agc gtg 2880 Leu Glu Ala Arg Val Ser Asp Phe Gly Met Ala Arg Leu MetSer Val 945 950 955 960 gtg gat aca cac ctt agc gtg tcc act ctt gct ggaacg cca ggg tat 2928 Val Asp Thr His Leu Ser Val Ser Thr Leu Ala Gly ThrPro Gly Tyr 965 970 975 gta cca ccg gag tac tac cag agc ttc aga tgc accacc aag ggt gat 2976 Val Pro Pro Glu Tyr Tyr Gln Ser Phe Arg Cys Thr ThrLys Gly Asp 980 985 990 gtt tat agc tat ggt gtt gtg ttg ctg gag ctg ctcacc ggg aaa ccg 3024 Val Tyr Ser Tyr Gly Val Val Leu Leu Glu Leu Leu ThrGly Lys Pro 995 1000 1005 ccg acg gac tcg gca gac ttt ggc gag gac aataac ctt gtg ggg 3069 Pro Thr Asp Ser Ala Asp Phe Gly Glu Asp Asn Asn LeuVal Gly 1010 1015 1020 tgg gtc aag cag cac acc aaa ttg aag atc acg gatgtc ttc gac 3114 Trp Val Lys Gln His Thr Lys Leu Lys Ile Thr Asp Val PheAsp 1025 1030 1035 cct gag cta ctc aag gag gat cca tcc gtc gag ctt gagctg ctg 3159 Pro Glu Leu Leu Lys Glu Asp Pro Ser Val Glu Leu Glu Leu Leu1040 1045 1050 gag cat ttg aaa atc gcc tgt gcg tgc ttg gat gac cgg ccgtcg 3204 Glu His Leu Lys Ile Ala Cys Ala Cys Leu Asp Asp Arg Pro Ser1055 1060 1065 agg cgg ccg acg atg ctg aag gtg atg gca atg ttc aag gagatc 3249 Arg Arg Pro Thr Met Leu Lys Val Met Ala Met Phe Lys Glu Ile1070 1075 1080 caa gct ggg tcg acg gtc gac tcg aag acc tcg tcg gcg gcagcg 3294 Gln Ala Gly Ser Thr Val Asp Ser Lys Thr Ser Ser Ala Ala Ala1085 1090 1095 ggc tcg atc gat gag gga ggc tat ggg gtc ctt gac atg cccctc 3339 Gly Ser Ile Asp Glu Gly Gly Tyr Gly Val Leu Asp Met Pro Leu1100 1105 1110 agg gaa gcc aag gag gag aag gat tagaaacaac aaccaccgac3383 Arg Glu Ala Lys Glu Glu Lys Asp 1115 1120 acacaggaga aacagccggcggtgagtggc caccaacgag gccagtcggc ggcgaaatgc 3443 ccgtagaaac aacagtcattcagaatcaga tggatgccat tttgaactct ccacacaagc 3503 ttagcaatcg cttctgatggtgctacaaga taagaatttt ccagctgtag gttgatcagt 3563 cgaagttgtt atgtacctataggagtagat cttttcttct ttcttttttc gcagctttct 3623 tcgtctccct gtttgtttttcccgtcgcgt cgcagtaaga gctgtgtatg tacatatata 3683 aatgttgaat tttctttggcgcaaaat 3710 2 1121 PRT Oryza sativa MISC_FEATURE (1)..(1121) BRI1protein (Genbank Accession BAB68053) 2 Met Asp Ser Leu Trp Ala Ala IleAla Ala Leu Phe Val Ala Ala Ala 1 5 10 15 Val Val Val Arg Gly Ala AlaAla Ala Asp Asp Ala Gln Leu Leu Glu 20 25 30 Glu Phe Arg Gln Ala Val ProAsn Gln Ala Ala Leu Lys Gly Trp Ser 35 40 45 Gly Gly Asp Gly Ala Cys ArgPhe Pro Gly Ala Gly Cys Arg Asn Gly 50 55 60 Arg Leu Thr Ser Leu Ser LeuAla Gly Val Pro Leu Asn Ala Glu Phe 65 70 75 80 Arg Ala Val Ala Ala ThrLeu Leu Gln Leu Gly Ser Val Glu Val Leu 85 90 95 Ser Leu Arg Gly Ala AsnVal Ser Gly Ala Leu Ser Ala Ala Gly Gly 100 105 110 Ala Arg Cys Gly SerLys Leu Gln Ala Leu Asp Leu Ser Gly Asn Ala 115 120 125 Ala Leu Arg GlySer Val Ala Asp Val Ala Ala Leu Ala Ser Ala Cys 130 135 140 Gly Gly LeuLys Thr Leu Asn Leu Ser Gly Asp Ala Val Gly Ala Ala 145 150 155 160 LysVal Gly Gly Gly Gly Gly Pro Gly Phe Ala Gly Leu Asp Ser Leu 165 170 175Asp Leu Ser Asn Asn Lys Ile Thr Asp Asp Ser Asp Leu Arg Trp Met 180 185190 Val Asp Ala Gly Val Gly Ala Val Arg Trp Leu Asp Leu Ala Leu Asn 195200 205 Arg Ile Ser Gly Val Pro Glu Phe Thr Asn Cys Ser Gly Leu Gln Tyr210 215 220 Leu Asp Leu Ser Gly Asn Leu Ile Val Gly Glu Val Pro Gly GlyAla 225 230 235 240 Leu Ser Asp Cys Arg Gly Leu Lys Val Leu Asn Leu SerPhe Asn His 245 250 255 Leu Ala Gly Val Phe Pro Pro Asp Ile Ala Gly LeuThr Ser Leu Asn 260 265 270 Ala Leu Asn Leu Ser Asn Asn Asn Phe Ser GlyGlu Leu Pro Gly Glu 275 280 285 Ala Phe Ala Lys Leu Gln Gln Leu Thr AlaLeu Ser Leu Ser Phe Asn 290 295 300 His Phe Asn Gly Ser Ile Pro Asp ThrVal Ala Ser Leu Pro Glu Leu 305 310 315 320 Gln Gln Leu Asp Leu Ser SerAsn Thr Phe Ser Gly Thr Ile Pro Ser 325 330 335 Ser Leu Cys Gln Asp ProAsn Ser Lys Leu His Leu Leu Tyr Leu Gln 340 345 350 Asn Asn Tyr Leu ThrGly Gly Ile Pro Asp Ala Val Ser Asn Cys Thr 355 360 365 Ser Leu Val SerLeu Asp Leu Ser Leu Asn Tyr Ile Asn Gly Ser Ile 370 375 380 Pro Ala SerLeu Gly Asp Leu Gly Asn Leu Gln Asp Leu Ile Leu Trp 385 390 395 400 GlnAsn Glu Leu Glu Gly Glu Ile Pro Ala Ser Leu Ser Arg Ile Gln 405 410 415Gly Leu Glu His Leu Ile Leu Asp Tyr Asn Gly Leu Thr Gly Ser Ile 420 425430 Pro Pro Glu Leu Ala Lys Cys Thr Lys Leu Asn Trp Ile Ser Leu Ala 435440 445 Ser Asn Arg Leu Ser Gly Pro Ile Pro Ser Trp Leu Gly Lys Leu Ser450 455 460 Tyr Leu Ala Ile Leu Lys Leu Ser Asn Asn Ser Phe Ser Gly ProIle 465 470 475 480 Pro Pro Glu Leu Gly Asp Cys Gln Ser Leu Val Trp LeuAsp Leu Asn 485 490 495 Ser Asn Gln Leu Asn Gly Ser Ile Pro Lys Glu LeuAla Lys Gln Ser 500 505 510 Gly Lys Met Asn Val Gly Leu Ile Val Gly ArgPro Tyr Val Tyr Leu 515 520 525 Arg Asn Asp Glu Leu Ser Ser Glu Cys ArgGly Lys Gly Ser Leu Leu 530 535 540 Glu Phe Thr Ser Ile Arg Pro Asp AspLeu Ser Arg Met Pro Ser Lys 545 550 555 560 Lys Leu Cys Asn Phe Thr ArgMet Tyr Val Gly Ser Thr Glu Tyr Thr 565 570 575 Phe Asn Lys Asn Gly SerMet Ile Phe Leu Asp Leu Ser Tyr Asn Gln 580 585 590 Leu Asp Ser Ala IlePro Gly Glu Leu Gly Asp Met Phe Tyr Leu Met 595 600 605 Ile Met Asn LeuGly His Asn Leu Leu Ser Gly Thr Ile Pro Ser Arg 610 615 620 Leu Ala GluAla Lys Lys Leu Ala Val Leu Asp Leu Ser Tyr Asn Gln 625 630 635 640 LeuGlu Gly Pro Ile Pro Asn Ser Phe Ser Ala Leu Ser Leu Ser Glu 645 650 655Ile Asn Leu Ser Asn Asn Gln Leu Asn Gly Thr Ile Pro Glu Leu Gly 660 665670 Ser Leu Ala Thr Phe Pro Lys Ser Gln Tyr Glu Asn Asn Thr Gly Leu 675680 685 Cys Gly Phe Pro Leu Pro Pro Cys Asp His Ser Ser Pro Arg Ser Ser690 695 700 Asn Asp His Gln Ser His Arg Arg Gln Ala Ser Met Ala Ser SerIle 705 710 715 720 Ala Met Gly Leu Leu Phe Ser Leu Phe Cys Ile Ile ValIle Ile Ile 725 730 735 Ala Ile Gly Ser Lys Arg Arg Arg Leu Lys Asn GluGlu Ala Ser Thr 740 745 750 Ser Arg Asp Ile Tyr Ile Asp Ser Arg Ser HisSer Ala Thr Met Asn 755 760 765 Ser Asp Trp Arg Gln Asn Leu Ser Gly ThrAsn Leu Leu Ser Ile Asn 770 775 780 Leu Ala Ala Phe Glu Lys Pro Leu GlnAsn Leu Thr Leu Ala Asp Leu 785 790 795 800 Val Glu Ala Thr Asn Gly PheHis Ile Ala Cys Gln Ile Gly Ser Gly 805 810 815 Gly Phe Gly Asp Val TyrLys Ala Gln Leu Lys Asp Gly Lys Val Val 820 825 830 Ala Ile Lys Lys LeuIle His Val Ser Gly Gln Gly Asp Arg Glu Phe 835 840 845 Thr Ala Glu MetGlu Thr Ile Gly Lys Ile Lys His Arg Asn Leu Val 850 855 860 Pro Leu LeuGly Tyr Cys Lys Ala Gly Glu Glu Arg Leu Leu Val Tyr 865 870 875 880 AspTyr Met Lys Phe Gly Ser Leu Glu Asp Val Leu His Asp Arg Lys 885 890 895Lys Ile Gly Lys Lys Leu Asn Trp Glu Ala Arg Arg Lys Ile Ala Val 900 905910 Gly Ala Ala Arg Gly Leu Ala Phe Leu His His Asn Cys Ile Pro His 915920 925 Ile Ile His Arg Asp Met Lys Ser Ser Asn Val Leu Ile Asp Glu Gln930 935 940 Leu Glu Ala Arg Val Ser Asp Phe Gly Met Ala Arg Leu Met SerVal 945 950 955 960 Val Asp Thr His Leu Ser Val Ser Thr Leu Ala Gly ThrPro Gly Tyr 965 970 975 Val Pro Pro Glu Tyr Tyr Gln Ser Phe Arg Cys ThrThr Lys Gly Asp 980 985 990 Val Tyr Ser Tyr Gly Val Val Leu Leu Glu LeuLeu Thr Gly Lys Pro 995 1000 1005 Pro Thr Asp Ser Ala Asp Phe Gly GluAsp Asn Asn Leu Val Gly 1010 1015 1020 Trp Val Lys Gln His Thr Lys LeuLys Ile Thr Asp Val Phe Asp 1025 1030 1035 Pro Glu Leu Leu Lys Glu AspPro Ser Val Glu Leu Glu Leu Leu 1040 1045 1050 Glu His Leu Lys Ile AlaCys Ala Cys Leu Asp Asp Arg Pro Ser 1055 1060 1065 Arg Arg Pro Thr MetLeu Lys Val Met Ala Met Phe Lys Glu Ile 1070 1075 1080 Gln Ala Gly SerThr Val Asp Ser Lys Thr Ser Ser Ala Ala Ala 1085 1090 1095 Gly Ser IleAsp Glu Gly Gly Tyr Gly Val Leu Asp Met Pro Leu 1100 1105 1110 Arg GluAla Lys Glu Glu Lys Asp 1115 1120 3 4538 DNA Oryza sativa misc_feature(1)..(4538) OsBRI1 genomic DNA 3 ctcgagcaga ccaccggctg tccccggccgtcggatcgga tcgggattta acctcgccgt 60 aagccgcgga taagcgcggg ggattagaatacttaacctc tcctctgctc tcctcctcac 120 ccggcttaag cgcgcggggg ggctgcgattcgcaggcgag agcacatgcc atgtgacccc 180 accccaccac tcctccctca cctacagctgttaaggccag tcacaatggg ggtttcactg 240 gtgtgtcatg cacatttaat aggggtaagactgaataaaa aatgattatt tgcatgaaat 300 ggggatgaga gagaaggaaa gagtttcatcctggtgaaac tcgtcagcgt cgtttccaag 360 tcctcggtaa cagagtgaaa cccccgttgaggccgattcg tttcattcac cggatctctt 420 gcgtccgcct ccgccgtgcg acctccgcattctcccgcgc cgcgccggat tttgggtaca 480 aatgatccca gcaacttgta tcaattaaatgctttgctta gtcttggaaa cgtcaaagtg 540 aaacccctcc actgtgggga ttgtttcataaaagatttca tttgagagaa gatggtataa 600 tattttgggt agccgtgcaa tgacactagccattgtgact ggcctaattg ctctccccct 660 ctactgtgtt tgcgtctttc ttcttcgcttctctttctct ctctctccat ctcctcctca 720 tcacttccca ctctccccct tctgtctctctactttctct ctctaccgcc gctctcgcag 780 caggccaggt tctctctaat ggtcgtgaggcagtgagctc gctcgtacat ggattccttg 840 tgggcagcga tagcggcact gtttgtggcggcggcggtgg tggtgagggg ggcggcggcg 900 gccgacgacg cccagctgct cgaggagttcaggcaggcgg tgccgaacca ggcggcgctc 960 aaggggtgga gcggcggcga cggcgcgtgcaggttcccgg gggccgggtg ccggaacggg 1020 aggctcacgt cgctgtcgct cgccggcgtgccgctcaatg ccgagttccg cgccgtcgcg 1080 gccaccctgc tgcagctcgg cagcgtcgaggtgctgagcc tccgcggcgc caacgtcagc 1140 ggcgcgctct cggcggctgg cggcgcgaggtgcgggagca agctgcaggc gctcgatttg 1200 tccgggaatg ccgcgctccg gggctccgtcgccgacgtgg cggccctggc cagcgcctgc 1260 ggcggcctca agacgctgaa tctctccggcgatgcggttg gtgcggcgaa ggtcggtggc 1320 ggtggtggcc cgggctttgc cgggctggactcgcttgatt tgtccaacaa caagatcacc 1380 gacgatagcg acctccggtg gatggtggatgccggagtcg gggcagtacg gtggttggac 1440 cttgccctga acaggatctc cggtgtcccggagttcacca actgctccgg gcttcagtac 1500 cttgacctct ccggcaacct catcgtcggtgaggtgcccg gcggggcact ttccgactgc 1560 cgcggtctga aagtgctcaa cctctccttcaaccacctcg ccggcgtgtt ccctccggac 1620 atcgccggcc tcacgtcgct caacgccctcaacctctcca acaacaactt ctccggcgag 1680 ctccccggcg aggctttcgc aaagctgcagcagcttacgg cgctctccct ctccttcaac 1740 cacttcaacg gctccatccc ggacaccgtagcctcgctgc cggagctcca gcagctcgac 1800 ctcagctcca acaccttctc cggcaccatcccgtcgtccc tctgccaaga tcccaactcc 1860 aagctccatc tgctgtacct tcagaacaactacctcaccg gcggcatccc agacgccgtc 1920 tccaactgca ccagcctcgt ctccctcgacctcagcctca actacatcaa tgggtccatc 1980 ccggcatccc tcggcgacct tggcaacctgcaggacctca tcctgtggca gaacgagctg 2040 gagggcgaga taccggcgtc cctgtcgcgcattcagggcc tcgagcatct catcctcgac 2100 tacaacgggc tcacgggtag catcccgccggagctagcca agtgcaccaa gctgaactgg 2160 atttctttgg cgagcaaccg gctgtccgggccaatccctt catggcttgg gaagctcagc 2220 tacttggcta tcttgaagct cagcaacaattccttctcgg ggcctatacc gccagagctc 2280 ggtgactgcc agagcttggt gtggctggacctgaatagca atcagctgaa tggatcaata 2340 cccaaagagc tggccaaaca gtctgggaagatgaatgttg gcctcatagt tggacggcct 2400 tacgtttatc ttcgcaacga cgagctgagcagcgagtgcc gtggcaaggg gagcttgctg 2460 gagtttacca gcatccgacc tgatgacctcagtcggatgc cgagcaagaa gctgtgcaac 2520 ttcacaagaa tgtatgtggg gagcacggagtacaccttca acaagaatgg ttcgatgata 2580 tttctcgatt tgtcatataa tcagctggactcggcgattc ctggcgagct gggggacatg 2640 ttctacctca tgatcatgaa tcttgggcacaacctactgt caggtaccat cccatcgcgg 2700 ctagcagagg ccaagaagct tgcggtgcttgacctgtcgt ataaccagtt ggaagggcca 2760 atacccaact ctttctcggc actttccttgtcggagatca atctgtcaaa taatcagctg 2820 aatggaacaa ttccagagct tggttcccttgccacatttc cgaagagcca gtatgagaat 2880 aacactggtt tatgtggctt cccactgccaccatgtgacc atagttcccc aagatcttcc 2940 aatgaccacc aatcccaccg gaggcaggcatcgatggcaa gcagtatcgc tatgggactg 3000 ttattctcac tgttctgtat aattgtgatcatcatagcca ttgggagcaa gcggcggagg 3060 ctgaagaatg aggaggcgag tacctctcgtgatatatata ttgatagcag gtcacattct 3120 gcaactatga attctgattg gaggcaaaatctctccggta caaatcttct tagcatcaac 3180 ctggctgcat tcgagaagcc attgcagaatctcaccctgg ctgatcttgt tgaggccaca 3240 aatggcttcc acatcgcatg ccaaattgggtctggtgggt ttggtgatgt ctacaaggca 3300 cagctcaagg atgggaaggt tgttgcaatcaagaagctaa tacatgtgag cgggcagggt 3360 gaccgggagt tcactgcaga aatggagaccattggcaaga tcaaacaccg taaccttgtt 3420 ccacttcttg gctattgcaa ggctggtgaggagcggttgt tggtgtatga ttacatgaag 3480 tttggcagct tggaggatgt gttgcatgaccgcaaaaaga tcggtaaaaa gctgaattgg 3540 gaggcaagac ggaaaatcgc tgttggagcagcaaggggtt tggcattcct ccaccacaat 3600 tgcattcctc acatcattca ccgagacatgaagtcgagca atgtgcttat cgatgaacaa 3660 ctggaagcaa gggtatctga tttcggtatggcgaggctga tgagcgtggt ggatacacac 3720 cttagcgtgt ccactcttgc tggaacgccagggtatgtac caccggagta ctaccagagc 3780 ttcagatgca ccaccaaggg tgatgtttatagctatggtg ttgtgttgct ggagctgctc 3840 accgggaaac cgccgacgga ctcggcagactttggcgagg acaataacct tgtggggtgg 3900 gtcaagcagc acaccaaatt gaagatcacggatgtcttcg accctgagct actcaaggag 3960 gatccatccg tcgagcttga gctgctggagcatttgaaaa tcgcctgtgc gtgcttggat 4020 gaccggccgt cgaggcggcc gacgatgctgaaggtgatgg caatgttcaa ggagatccaa 4080 gctgggtcga cggtcgactc gaagacctcgtcggcggcag cgggctcgat cgatgaggga 4140 ggctatgggg tccttgacat gcccctcagggaagccaagg aggagaagga ttagaaacaa 4200 caaccaccga cacacaggag aaacagccggcggtgagtgg ccaccaacga ggccagtcgg 4260 cggcgaaatg cccgtagaaa caacagtcattcagaatcag atggatgcca ttttgaactc 4320 tccacacaag cttagcaatc gcttctgatggtgctacaag ataagaattt tccagctgta 4380 ggttgatcag tcgaagttgt tatgtacctataggagtaga tcttttcttc tttctttttt 4440 cgcagctttc ttcgtctccc tgtttgtttttcccgtcgcg tcgcagtaag agctgtgtat 4500 gtacatatat aaatgttgaa ttttctttggcgcaaaat 4538 4 38 DNA Artificial Sequence 5′ primer sequence 4ggctctagac agccatggcg agcaagcggc ggaggctg 38 5 19 DNA ArtificialSequence 3′ primer sequence 5 agatctactc ctataggta 19 6 1196 PRTArabidopsis thaliana MISC_FEATURE (1)..(1196) BRI1 protein (GenbankAccession NP_195650) 6 Met Lys Thr Phe Ser Ser Phe Phe Leu Ser Val ThrThr Leu Phe Phe 1 5 10 15 Phe Ser Phe Phe Ser Leu Ser Phe Gln Ala SerPro Ser Gln Ser Leu 20 25 30 Tyr Arg Glu Ile His Gln Leu Ile Ser Phe LysAsp Val Leu Pro Asp 35 40 45 Lys Asn Leu Leu Pro Asp Trp Ser Ser Asn LysAsn Pro Cys Thr Phe 50 55 60 Asp Gly Val Thr Cys Arg Asp Asp Lys Val ThrSer Ile Asp Leu Ser 65 70 75 80 Ser Lys Pro Leu Asn Val Gly Phe Ser AlaVal Ser Ser Ser Leu Leu 85 90 95 Ser Leu Thr Gly Leu Glu Ser Leu Phe LeuSer Asn Ser His Ile Asn 100 105 110 Gly Ser Val Ser Gly Phe Lys Cys SerAla Ser Leu Thr Ser Leu Asp 115 120 125 Leu Ser Arg Asn Ser Leu Ser GlyPro Val Thr Thr Leu Thr Ser Leu 130 135 140 Gly Ser Cys Ser Gly Leu LysPhe Leu Asn Val Ser Ser Asn Thr Leu 145 150 155 160 Asp Phe Pro Gly LysVal Ser Gly Gly Leu Lys Leu Asn Ser Leu Glu 165 170 175 Val Leu Asp LeuSer Ala Asn Ser Ile Ser Gly Ala Asn Val Val Gly 180 185 190 Trp Val LeuSer Asp Gly Cys Gly Glu Leu Lys His Leu Ala Ile Ser 195 200 205 Gly AsnLys Ile Ser Gly Asp Val Asp Val Ser Arg Cys Val Asn Leu 210 215 220 GluPhe Leu Asp Val Ser Ser Asn Asn Phe Ser Thr Gly Ile Pro Phe 225 230 235240 Leu Gly Asp Cys Ser Ala Leu Gln His Leu Asp Ile Ser Gly Asn Lys 245250 255 Leu Ser Gly Asp Phe Ser Arg Ala Ile Ser Thr Cys Thr Glu Leu Lys260 265 270 Leu Leu Asn Ile Ser Ser Asn Gln Phe Val Gly Pro Ile Pro ProLeu 275 280 285 Pro Leu Lys Ser Leu Gln Tyr Leu Ser Leu Ala Glu Asn LysPhe Thr 290 295 300 Gly Glu Ile Pro Asp Phe Leu Ser Gly Ala Cys Asp ThrLeu Thr Gly 305 310 315 320 Leu Asp Leu Ser Gly Asn His Phe Tyr Gly AlaVal Pro Pro Phe Phe 325 330 335 Gly Ser Cys Ser Leu Leu Glu Ser Leu AlaLeu Ser Ser Asn Asn Phe 340 345 350 Ser Gly Glu Leu Pro Met Asp Thr LeuLeu Lys Met Arg Gly Leu Lys 355 360 365 Val Leu Asp Leu Ser Phe Asn GluPhe Ser Gly Glu Leu Pro Glu Ser 370 375 380 Leu Thr Asn Leu Ser Ala SerLeu Leu Thr Leu Asp Leu Ser Ser Asn 385 390 395 400 Asn Phe Ser Gly ProIle Leu Pro Asn Leu Cys Gln Asn Pro Lys Asn 405 410 415 Thr Leu Gln GluLeu Tyr Leu Gln Asn Asn Gly Phe Thr Gly Lys Ile 420 425 430 Pro Pro ThrLeu Ser Asn Cys Ser Glu Leu Val Ser Leu His Leu Ser 435 440 445 Phe AsnTyr Leu Ser Gly Thr Ile Pro Ser Ser Leu Gly Ser Leu Ser 450 455 460 LysLeu Arg Asp Leu Lys Leu Trp Leu Asn Met Leu Glu Gly Glu Ile 465 470 475480 Pro Gln Glu Leu Met Tyr Val Lys Thr Leu Glu Thr Leu Ile Leu Asp 485490 495 Phe Asn Asp Leu Thr Gly Glu Ile Pro Ser Gly Leu Ser Asn Cys Thr500 505 510 Asn Leu Asn Trp Ile Ser Leu Ser Asn Asn Arg Leu Thr Gly GluIle 515 520 525 Pro Lys Trp Ile Gly Arg Leu Glu Asn Leu Ala Ile Leu LysLeu Ser 530 535 540 Asn Asn Ser Phe Ser Gly Asn Ile Pro Ala Glu Leu GlyAsp Cys Arg 545 550 555 560 Ser Leu Ile Trp Leu Asp Leu Asn Thr Asn LeuPhe Asn Gly Thr Ile 565 570 575 Pro Ala Ala Met Phe Lys Gln Ser Gly LysIle Ala Ala Asn Phe Ile 580 585 590 Ala Gly Lys Arg Tyr Val Tyr Ile LysAsn Asp Gly Met Lys Lys Glu 595 600 605 Cys His Gly Ala Gly Asn Leu LeuGlu Phe Gln Gly Ile Arg Ser Glu 610 615 620 Gln Leu Asn Arg Leu Ser ThrArg Asn Pro Cys Asn Ile Thr Ser Arg 625 630 635 640 Val Tyr Gly Gly HisThr Ser Pro Thr Phe Asp Asn Asn Gly Ser Met 645 650 655 Met Phe Leu AspMet Ser Tyr Asn Met Leu Ser Gly Tyr Ile Pro Lys 660 665 670 Glu Ile GlySer Met Pro Tyr Leu Phe Ile Leu Asn Leu Gly His Asn 675 680 685 Asp IleSer Gly Ser Ile Pro Asp Glu Val Gly Asp Leu Arg Gly Leu 690 695 700 AsnIle Leu Asp Leu Ser Ser Asn Lys Leu Asp Gly Arg Ile Pro Gln 705 710 715720 Ala Met Ser Ala Leu Thr Met Leu Thr Glu Ile Asp Leu Ser Asn Asn 725730 735 Asn Leu Ser Gly Pro Ile Pro Glu Met Gly Gln Phe Glu Thr Phe Pro740 745 750 Pro Ala Lys Phe Leu Asn Asn Pro Gly Leu Cys Gly Tyr Pro LeuPro 755 760 765 Arg Cys Asp Pro Ser Asn Ala Asp Gly Tyr Ala His His GlnArg Ser 770 775 780 His Gly Arg Arg Pro Ala Ser Leu Ala Gly Ser Val AlaMet Gly Leu 785 790 795 800 Leu Phe Ser Phe Val Cys Ile Phe Gly Leu IleLeu Val Gly Arg Glu 805 810 815 Met Arg Lys Arg Arg Arg Lys Lys Glu AlaGlu Leu Glu Met Tyr Ala 820 825 830 Glu Gly His Gly Asn Ser Gly Asp ArgThr Ala Asn Asn Thr Asn Trp 835 840 845 Lys Leu Thr Gly Val Lys Glu AlaLeu Ser Ile Asn Leu Ala Ala Phe 850 855 860 Glu Lys Pro Leu Arg Lys LeuThr Phe Ala Asp Leu Leu Gln Ala Thr 865 870 875 880 Asn Gly Phe His AsnAsp Ser Leu Ile Gly Ser Gly Gly Phe Gly Asp 885 890 895 Val Tyr Lys AlaIle Leu Lys Asp Gly Ser Ala Val Ala Ile Lys Lys 900 905 910 Leu Ile HisVal Ser Gly Gln Gly Asp Arg Glu Phe Met Ala Glu Met 915 920 925 Glu ThrIle Gly Lys Ile Lys His Arg Asn Leu Val Pro Leu Leu Gly 930 935 940 TyrCys Lys Val Gly Asp Glu Arg Leu Leu Val Tyr Glu Phe Met Lys 945 950 955960 Tyr Gly Ser Leu Glu Asp Val Leu His Asp Pro Lys Lys Ala Gly Val 965970 975 Lys Leu Asn Trp Ser Thr Arg Arg Lys Ile Ala Ile Gly Ser Ala Arg980 985 990 Gly Leu Ala Phe Leu His His Asn Cys Ser Pro His Ile Ile HisArg 995 1000 1005 Asp Met Lys Ser Ser Asn Val Leu Leu Asp Glu Asn LeuGlu Ala 1010 1015 1020 Arg Val Ser Asp Phe Gly Met Ala Arg Leu Met SerAla Met Asp 1025 1030 1035 Thr His Leu Ser Val Ser Thr Leu Ala Gly ThrPro Gly Tyr Val 1040 1045 1050 Pro Pro Glu Tyr Tyr Gln Ser Phe Arg CysSer Thr Lys Gly Asp 1055 1060 1065 Val Tyr Ser Tyr Gly Val Val Leu LeuGlu Leu Leu Thr Gly Lys 1070 1075 1080 Arg Pro Thr Asp Ser Pro Asp PheGly Asp Asn Asn Leu Val Gly 1085 1090 1095 Trp Val Lys Gln His Ala LysLeu Arg Ile Ser Asp Val Phe Asp 1100 1105 1110 Pro Glu Leu Met Lys GluAsp Pro Ala Leu Glu Ile Glu Leu Leu 1115 1120 1125 Gln His Leu Lys ValAla Val Ala Cys Leu Asp Asp Arg Ala Trp 1130 1135 1140 Arg Arg Pro ThrMet Val Gln Val Met Ala Met Phe Lys Glu Ile 1145 1150 1155 Gln Ala GlySer Gly Ile Asp Ser Gln Ser Thr Ile Arg Ser Ile 1160 1165 1170 Glu AspGly Gly Phe Ser Thr Ile Glu Met Val Asp Met Ser Ile 1175 1180 1185 LysGlu Val Pro Glu Gly Lys Leu 1190 1195

1. A DNA encoding a protein comprising the amino acid sequence of SEQ IDNO:
 2. 2. The DNA of claim 1, wherein the DNA is a cDNA or a genomicDNA.
 3. The DNA of claim 1, wherein the DNA comprises a coding region ofthe nucleotide sequence of SEQ ID NO: 1 or
 3. 4. A DNA encoding aprotein which has 55% or more homology to the amino acid sequence of SEQID NO: 2 and which is functionally equivalent to a protein comprisingthe amino acid sequence of SEQ ID NO: 2, said DNA being selected fromthe group consisting of (a) a DNA encoding a protein comprising theamino acid sequence of SEQ ID NO: 2 in which one or more amino acids aresubstituted, deleted, added, and/or inserted; and (b) a DNA hybridizingunder stringent conditions with a DNA comprising the nucleotide sequenceof SEQ ID NO: 1 or
 3. 5. The DNA of claim 4, wherein the DNA encodes aprotein having a function selected from the group consisting of afunction of increasing brassinosteroid sensitivity in a plant, afunction of inducing elongation of internode cells of a stem of a plant,a function of positioning microtubules perpendicular to the direction ofelongation in an internode of a stem of a plant, a function ofsuppressing elongation of an internode of a neck of a plant, and afunction of increasing inclination of a lamina of a plant.
 6. The DNA ofclaim 4 or 5, wherein the DNA is derived from a monocotyledonous plant.7. The DNA of claim 6, wherein the DNA is derived from a plant of theGramineae family.
 8. A DNA encoding an antisense RNA complementary to atranscript of the DNA of any one of claims 1 to
 7. 9. A DNA encoding anRNA having ribozyme activity which specifically cleaves a transcript ofthe DNA of any one of claims 1 to
 7. 10. A DNA which encodes an RNArepressing expression of the DNA of any one of claims 1 to 7 due toco-suppression when expressed in a plant cell and which has 90% or morehomology to the DNA of any one of claims 1 to
 7. 11. A DNA which encodesa protein having a dominant negative phenotype to that of a proteinencoded by the DNA of any one of claims 1 to
 7. 12. A vector whichcomprises the DNA of any one of claims 1 to
 7. 13. A transformed cellwhich comprises the DNA of any one of claims 1 to 7 or the vector ofclaim
 12. 14. A protein encoded by the DNA of any one of claims 1 to 7.15. A method for producing the protein of claim 14, the methodcomprising the steps of culturing the transformed cell of claim 13 andrecovering an expressed protein from said transformed cell or a culturesupernatant thereof.
 16. A vector comprising the DNA of any one ofclaims 8 to
 11. 17. A transformed plant cell comprising the DNA of anyone of claims 1 to 11 or the vector of claim 12 or
 16. 18. A transformedplant comprising the transformed plant cell of claim
 17. 19. Atransformed plant which is a progeny or a clone of the transformed plantof claim
 18. 20. A breeding material of the transformed plant of claim18 or
 19. 21. An antibody which binds to the protein of claim 14.